Alpha-aminoisobutyric Acid Transport Research Articles

Effect of increased glucose load on maternal-fetal transport of alpha-aminoisobutyric acid in the perfused human placenta: in vitro study.

The role of hyperglycemia on modulation of maternal-fetal transport of amino acids in humans is little understood. Hence, we have explored the effect of increased glucose load on transport kinetics of a model non-metabolizable amino acid, alpha-aminoisobutyric acid (AIB), in the human placenta in vitro. Transport kinetics of AIB in maternal-fetal direction was studied using perfusion of isolated human placental lobules. NCTC (National Culture and Tissue Collection)-135 medium, diluted with Earle's buffered salt solution was used as the perfusate and tritiated water was used as the reference marker. Effect of increased glucose load on transport kinetics of study and reference substances was studied in normal term placentae (n=5; gestational age, 38.5 +/- 0.5 weeks) in succeeding experimental phases, after a control perfusion phase with physiological glucose concentration. AIB transport fraction (TF), relative to tritiated water TF, averaged 54.8% in control euglycemic phase while in hyperglycemic concentration phases of 27.8 and 55.6 mM, the AIB TF index averaged 42.4% and 38.2%, respectively. Analysis of variance revealed that the difference was statistically significant. Similarly, absorption rate index of the amino acid was also significantly lower in the hyperglycemic perfusion phases compared to control euglycemic phase. We conclude that hyperglycemia may play a deleterious role in limiting maternal-fetal transport of A-type amino acids in the in vivo state.

Amino acid transport by intestinal brush border vesicles of a marine fish, Boops salpa

The transport of glycine, alanine, methionine and alpha amino-isobutyric acid (AIB) was studied on brush border membrane vesicles of Boops salpa, a marine fish. This transport was Na +-, Cl −- and pH-dependent. In the presence of NaCl, the uptake decreased as the pH increased from 5.5 to 8.5. With Na 2SO 4, the transport of the four amino acids was strongly reduced and the pH optimum was 7–8. In the presence of NaCl, amino acid transport was described by high and low affinity kinetics. The K t of the high-affinity component was comparable for glycine, alanine and methionine (0.1 mM), and was significantly enhanced for AIB (0.6 mM). The J max of the low affinity component was significantly lower for methionine and AIB than for glycine and alanine. Lowering the sodium concentration from 80 to 20 mM significantly increased K t and J max of the high-affinity component of glycine transport. Moreover, the kinetics of AIB transport under 100 mM Na + were similar to glycine kinetics under 40 mM Na + and the two amino acids competed for the same carrier(s). These results suggest that chloride ions are essential in neutral amino acid transport in Boops, that multiple saturable components are involved in this process, and that sodium plays an important role in the differences between the transport kinetics of amino acids.

Photosensitization of cells with different metastatic potentials by liposome-delivered Zn(II)-phthalocyanine

The phototoxicity of liposome-incorporated Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) was studied in the tumorigenic but nonmetastatic (RE4) and the highly metastatic (4R) transformed rat embryo fibroblasts. Upon irradiation with 585-605 nm light in the presence of ZnPc, the cell survival drastically decreased, while it was unaffected by ZnPcTS. Enzymatic assays showed that ZnPc induced about a 60% decrease in the activity of the mitochondrial enzymes NADH and succinate dehydrogenase after 3 min of irradiation, while no significant reduction in the activity of lactate dehydrogenase and lysosomal N-acetyl-beta-glucosaminidase was observed. The transport of thymidine, deoxyglucose and alpha-aminoisobutyric acid through the plasma membrane was strongly inhibited after irradiation. Similarly, the intracellular ATP content was significantly reduced. The reduction of DNA biosynthesis showed a time dependence quite similar to the photo-induced decrease in cell survival. No repair of cellular functions affected by ZnPc was observed in the 2 cell lines. These results indicate that, under our experimental conditions, hydrophobic ZnPc exerts its cytotoxic activity mainly by impairing those functions localized in the plasma membrane of the cells.

Changes in the permeability of the blood-brain barrier following sodium dodecyl sulphate administration in the rat.

The blood-brain barrier (BBB) arises from epithelial-like tight junctions that virtually cement adjoining capillary endothelium together in the brain microvasculature. Several experimental manipulations have been shown able to increase the permeability of brain capillaries, by altering endothelial cell membrane integrity or activating specific biochemical pathways involved in regulation of BBB functionality. Because of its amphiphilic nature, sodium dodecyl sulphate (an anionic surfactant widely used as solubilizer or stabilizer in several pharmaceutical preparations; SDS) may enter into interactions with the major membrane components, which are lipids and proteins. The aim of the present study was to determine the effect of an intracarotid infusion of SDS (25, 50 and 100 microg/kg; infusion rate: 3 ml/min for 30 s) on the functionality of the BBB in the rat. An extensive, dose-dependent Evans blue extravasation was observed, in the ipsilateral brain hemisphere, 15 min following SDS infusion. These results were confirmed by the significant increase in [14C]alpha-aminoisobutyric acid ([14C]AIB) transport (evaluated by calculating a unidirectional transfer constant, Ki, for the tracer from blood to brain) measured in several ipsilateral brain regions 2 min after SDS infusion; this SDS-elicited BBB opening to [14C]AIB proved to be reversible. Since the BBB is created by the plasma membrane and tight junctions of the endothelial cells, the change in BBB permeability caused by SDS might be explained as a nonspecific surfactant-membrane interaction. Furthermore, SDS might affect the functional characteristics of brain vascular endothelial cells by an interaction with specific BBB proteins and/or biochemical pathways. In conclusion, one can suggest that intracarotid infusion of SDS might provide a useful clinical approach for the intentional introduction of different substances into the brain. On the other hand, these findings should call attention to possible dangerous consequences of using SDS as solubilizer in drug excipients.

Gastric fundic inhibition of sugar transport across the intestinal mucosa of guinea-pig.

A low molecular weight peptide designated gastric fundic factor (GFF), extracted from porcine fundic mucosa and administered to the serosal surface of mucosal sheets from guinea-pig intestine, decreased the transport of luminal glucose across the sheets by up to 70%. The results show that gastric fundic inhibition of glucose absorption observed in different animal models in vivo can be reproduced in vitro, and suggest that the intestinal mucosa itself is the target for peptide hormone(s) released by the gastric fundic mucosa. Simultaneous transport of alpha-aminoisobutyric acid, a nonmetabolisable amino acid, through the jejunal mucosa was unaffected as was paracellular permeation by inulin. However, amino acid transport was also reduced when GFF was administered to sheets of ileal mucosa. The intestinal mucosal sheet in vitro is a sensitive and convenient model with which to follow the purification of GFF to homogeneity.

Blood-brain barrier permeability changes after experimental subarachnoid hemorrhage.

Basic mechanisms underlying cerebrovascular permeability responses to subarachnoid hemorrhage (SAH) are still to be defined in detail. Previous investigations examining the occurrence of blood-brain barrier (BBB) breakdown after SAH in the experimental setting have yielded conflicting results. In a rat model of SAH, we assessed BBB changes by means of the quantitative [14C]-alpha-aminoisobutyric acid technique. Experiments were carried out on the second day post-SAH. In blood-injected rats [14C]-alpha-aminoisobutyric acid transport across the BBB increased significantly in cerebral cortices and cerebellar gray matter, averaging 1.3 to 1.5 times control values. The present data indicate that SAH induces well-defined changes in BBB function, possibly involved in the pathogenesis of post-SAH cerebral dysfunction in humans. Results reported here have also potential clinical implications for the management of aneurysm patients.

Evidence for an essential sulfhydryl group at the substrate binding site of the A-system transporter of Ehrlich cell plasma membranes

Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na(+)-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na(+)-dependent alpha-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained approximately 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 microM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 microM p-chloromercuribenzenesulfonic acid inhibited Na(+)-dependent alpha-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane Protein Changes in an L1210 Leukemia Cell Line with a Translocation Defect in the Methotrexate-Tetrahydrofolate Cofactor Transport Carrier

We report on membrane protein changes in an L1210 leukemia cell line with a highly specific defect in the function of the methotrexate (MTX)-tetrahydrofolate cofactor transport carrier. This clonal line, MTXrA, made 100-fold resistant to MTX, was derived in a single step and exhibited stable resistance over 120 generations in the absence of drug. The transport defect was associated with a 10-fold decrease in influx Vmax without a change in influx Km. There was no difference between the MTXrA and parent lines in the levels or affinities of specific cell surface binders for MTX nor in the labeling of the 44-kDa membrane protein upon treatment with the specific affinity label, N-hydroxysuccinimide ester of tritiated MTX. Consistent with impaired carrier function was the observation that trans-stimulation of MTX influx by intracellular 5-formyltetrahydrofolate observed in the parent line was not demonstrated in the MTXrA line. The transport defect was highly specific for the MTX-tetrahydrofolate cofactor transport carrier. Initial uptake rates for 5-fluoro-2'-deoxyuridine and 2-deoxyglucose were unchanged and influx and net transport of alpha-aminoisobutyric acid were, in fact, increased. There was no cross-resistance of this line to phenylalanine mustard or cytosine arabinoside, agents that utilize specific amino acid and nucleoside transport carriers, respectively. SDS-polyacrylamide gel electrophoresis of purified plasma membrane preparations stained with Coomassie Blue revealed several protein differences between the parental and MTXrA lines. Most prominent is a band at approximately 190 kDa which ran with slightly greater mobility than a lesser staining band in the parent line. [3H]Borohydride labeling of cells also identified a distinct protein peak in the MTXrA line at approximately 190 kDa eliminated by prior treatment of cells with neuraminidase. Absence of expression of protein or mRNA related to the multidrug resistance gene as well as lack of cross-resistance to daunorubicin or trimetrexate indicate that this mechanism of resistance to MTX is completely unrelated to the multidrug resistance phenomenon observed with high molecular weight heterocyclic compounds. These data represent the first demonstration of membrane protein differences in a highly resistant L1210 murine leukemia cell line with a marked unique defect in MTX transport which appears to be related to impaired mobility of the tetrahydrofolate-cofactor carrier. Further studies are now required to elucidate the possible role of one or more of these proteins in the transport defect.

Effect of dimethyl sulfoxide on blood-to-brain transfer of α-aminoisobutyric acid: Examination of regional blood-brain barrier function

The effect of dimethyl sulfoxide (DMSO) on brain capillary permeability has been controversial. We have studied the effect of DMSO on unidirectional transport of alpha-aminoisobutyric acid (AIB) across the blood-brain barrier (BBB) in rats. Rats were treated with 15% DMSO intraperitoneally (i.p.), intravenously (i.v.) or by an i.p. injection in combination with an i.v. injection, or in some cases intra-arterially by rapid infusion into left external carotid artery. The unidirectional blood-to-brain transfer constant (Ki) for AIB was measured in each group after the animals were killed. DMSO administration did not significantly increase Ki as compared to control Kj. These results show that it is unlikely that DMSO increases the permeability of BBB and therefore do not support the proposal that DMSO can act as a carrier at the BBB for compounds with restricted vascular permeability.

The acute effects of growth hormone on amino acid transport and protein synthesis are due to its insulin-like action.

GH has acute stimulatory effects on amino acid transport and protein synthesis in a variety of tissues, but it has not been established whether these effects are expressions of the growth-promoting property of GH or of its separate insulin-like action. The 20,000-dalton structural variant of human GH (20K hGH) has been shown to have a high ratio of growth-promoting to insulin-like activity compared to native hGH (22K hGH), suggesting that it could be used as a tool to address the above question. Therefore, experiments were conducted to compare the relative abilities of native 22K hGH and 20K hGH, when added in vitro, to stimulate amino acid transport and protein synthesis in the isolated diaphragm of the female hypophysectomized rat. Paired intact hemidiaphragms were preincubated for 1 h in the absence or presence of various concentrations of 22K or 20K hGH. Then, 3-O-[14C]methylglucose was added to the medium to measure sugar transport as a test of insulin-like activity, and either alpha-[3H]aminoisobutyric acid acid or [3H] phenylalanine was also added to measure amino acid transport or protein synthesis, respectively, during a final hour of incubation. When the responses to the various concentrations of 22K and 20K were compared, 20K hGH was only about 20% as effective as 22K in stimulating 3-O-methylglucose transport, reflecting its markedly attenuated insulin-like activity on the diaphragm. Similarly, 20K hGH was only 20% as effective as 22K hGH in stimulating alpha-aminoisobutyric acid transport and phenylalanine incorporation into protein in the same muscles. Therefore, these findings support the idea that the rapid stimulatory effects of GH on amino acid transport and protein synthesis are expressions of the insulin-like action of GH and are not components of the response of target cells to its growth-promoting action.

The absence of γ‐glutamyltransferase activity in transport‐dependent methotrexate‐resistant hepatoma cells

A cell line derived from H35 hepatoma cells resistant to methotrexate (MTX) as a result of a defective transport system for MTX has been examined to determine how closely the variant resembles the parent cells with regard to other biochemical properties. The capacity of extracts of resistant cells to catalyze the poly-gamma-glutamylation of MTX was approximately twice as great as that of wild-type cell extracts. Evidence of similarity between wild-type and H35 R0.3 cells was derived from the equitoxic activity to both cell lines of nonclassical antifolates and other miscellaneous antineoplastics which act by a variety of mechanisms. Two phenotypic markers of hepatic cell function, alpha-aminoisobutyric acid (AIB) transport and tyrosine aminotransferase (TAT) activity inducibility, were present in both cell types, demonstrating the maintenance of these phenotypic properties in the H35 R0.3 cells. gamma-Glutamyltransferase (GGT, EC 2.3.2.2) activity differed in that it was present in wild-type cells and barely detectable in H35 R0.3 cells. The GGT activity reappeared in the H35 cells when they regained MTX sensitivity after incubation for 14-20 weeks in MTX-free media. Although defective MTX transport appeared to be correlated with the disappearance of GGT activity in an H35 variant cell line, no functional relationship between them is apparent at this time. It is possible that a lack of GGT activity may be evidence of a more differentiated phenotype in the transport-resistant cell line.

Effect of triiodothyronine on system A amino acid transport in cells of rat submandibular gland.

The effect of L-3,5,3'-triiodothyronine (T3) on alpha-aminoisobutyric acid (AIB) transport in isolated cell suspensions of rat submandibular gland was investigated. The uptake of ATB by these cells appeared to require extracellular Na+ and was inhibited by ouabain (10(-3) M). Cell suspensions from thyroidectomized rats which have been given three successive doses of T3 on alternate days (50 micrograms/100 g BW) showed a significant increase in AIB uptake compared with cells isolated from thyroidectomized rats treated with diluent. Efflux of AIB from the cell suspension was not affected by T3. There was no significant changes in AIB uptake 12 h after a single injection of T3 (50 micrograms/100 g BW). However, there was a significant 49% and 65% increase in AIB net uptake at 24 and 48 h, respectively, after T3 treatment. Under similar conditions, the cell suspension showed a 48% increase in NaK-ATPase activity at 12 h and to a peak of 61% at 24 h. Therefore, changes in NaK-ATPase activity preceded the changes in AIB net uptake upon treatment with T3, implying that AIB uptake is probably mediated, at least in part, by the activity of NaK-ATPase.

Glycogen depletion and increased insulin sensitivity and responsiveness in muscle after exercise.

As judged by its ability to stimulate glucose uptake and alpha-aminoisobutyric acid (AIB) transport, the sensitivity and the responsiveness of perfused rat muscle to insulin are enhanced after moderately intense treadmill exercise. In fed rats, these enhanced effects of insulin are predominantly restricted to muscles that performed work as evidenced by glycogen depletion. The present study was designed to examine the relationship between glycogen depletion per se and the postexercise changes in insulin action. Toward this end, fed and 48-h starved rats were run on a treadmill for 45 min at moderate intensity, and glucose and AIB uptake were then assessed using the isolated perfused hindquarter preparation. Glycogen is depleted in red muscles such as the soleus and red fibers of the gastrocnemius in fed rats immediately after such exercise, whereas, in starved rats, muscle glycogen is unchanged. As previously shown, the stimulation by insulin of glucose utilization by the hindquarter and AIB transport into red muscles was substantially increased in fed rats after the treadmill run. This was due to increases in both insulin sensitivity and responsiveness. In starved rats, the treadmill run also enhanced the ability of insulin to stimulate these processes; however, this was solely due to an increase in insulin sensitivity. No change in insulin responsiveness was observed. The results indicate that the enhanced sensitivity of muscle to insulin after exercise is not dependent on glycogen depletion, whereas increased insulin responsiveness does not occur in its absence. They also suggest that the mechanisms by which prior exercise acts to increase insulin sensitivity and responsiveness are different.

Insulin and exercise stimulate muscle alpha-aminoisobutyric acid transport by a Na +K +-ATPase independent pathway

Sodium ions are required for the active transport of amino acids such as alpha-aminoisobutyric acid (AIB) into skeletal muscle. To examine the role of Na +K +-ATPase in this phenomenon, studies were carried out using the isolated perfused rat hindquarter preparation. Perfusion for 30 min with ouabain at a dose sufficient to inhibit the Na +K + pump (10 −4M) inhibited the basal rate of AIB uptake in all muscles studied by up to 80%. However, it failed to inhibit the stimulation of AIB uptake, either by insulin (200 uU/ml) or electrically-induced muscle contractions. The increase in K+ release by the hindquarter in the presence of ouabain was the same under all conditions suggesting comparable inhibition of the Na +K + pump. These studies suggest that the basal, but not insulin or exercise-stimulated AIB transport into muscle is acutely dependent on a functional Na +K + pump. They also suggest that stimulated and basal uptake of AIB involve different mechanisms.

Binding, degradation, and biological activity of insulin in vascular smooth muscle cells.

The interaction of insulin with the vascular smooth muscle was studied using cultures derived from the bovine aortic arch. The cultured cells exhibited specific binding of 125I-insulin that was reversible and dependent on pH. Both insulin and insulinlike growth factor (IGF) I competed for 125I-insulin binding; IGF I, however, was less effective than insulin by at least an order of magnitude. Insulin binding was accompanied by internalization and degradation of the hormone in a temperature- and time-dependent manner. Chloroquine and other lysosomotropic agents elevated the internalized insulin and reduced its degradation. Pre-exposure of cell cultures to insulin resulted in downregulation of cell surface receptors. Insulin stimulated alpha-aminoisobutyric acid transport in confluent smooth muscle cells. The maximal response was observed at 100 ng/ml insulin with a half-maximal effect at 10 ng/ml. Sparse, serum-starved smooth muscle cells responded to insulin with a dose-dependent increase in [3H]-thymidine incorporation into DNA. Although the effect was already apparent at 1 ng/ml insulin, it reached near maximal level only at 10,000 ng/ml. IGF I also stimulated DNA synthesis in smooth muscle cells; however, at low concentrations insulin was more efficient in this respect. Human growth hormone was inactive. The data indicate the presence of specific receptors for insulin in bovine aortic smooth muscle cells. These receptors appear to mediate the metabolic activity as well as part of the mitogenic effect of insulin in these cells.

Muscle alpha-aminoisobutyric acid transport after exercise: enhanced stimulation by insulin.

After exercise the ability of insulin to stimulate glucose transport and glycogen synthesis in rat skeletal muscle is markedly enhanced (25). The present study was designed to determine whether prior exercise augments the stimulation of other processes by insulin and, if so, whether this can be attributed to an increase in insulin binding to its receptor. Toward this end rats were run on a treadmill for 45 min at moderate intensity and the uptake of alpha-aminoisobutyric acid (AIB) by muscle was then assessed using the isolated perfused hindquarter preparation. Approximately 30 min after the cessation of exercise, both the sensitivity and responsiveness of insulin-stimulated AIB uptake were significantly enhanced in the soleus and the red portion of the gastrocnemius. As previously shown for glucose transport and glycogen synthesis, only small effects were observed in the white portion of the gastrocnemius, which unlike the other muscles was not depleted of glycogen during the run. Insulin-stimulated glucose utilization was also enhanced in the incubated soleus muscle of exercised rats; however, insulin binding to the soleus was not altered. These studies indicate that the ability of insulin to stimulate processes other than glucose transport and glycogen synthesis is enhanced in skeletal muscle after exercise and that this is not due to an alteration in insulin binding. The changes in insulin-stimulated AIB uptake and glucose metabolism after exercise are the reverse of those found in denervated and immobilized muscle and in both situations insulin binding is not altered. This suggests that a common factor(s) modulates the increase in insulin effect after exercise and the insulin resistance of disuse.

L-Triiodothyronine (T3) stimulates growth of cultured GC cells by action early in the G1 period: evidence for mediation by the nuclear T3 receptor.

Incubation with T3 results in a dose-dependent increase in growth rate of cultured GC cells, a GH-producing rat pituitary tumor cell line. The T3-induced increase in growth rate results mainly from shortening of the G1 period from 79.4 +/- 4.3 (SD) h in cells grown in T3-depleted medium (-T3) to 10.0 +/- 0.9 h. This effect can also be demonstrated in synchronized populations. Addition of T3 (0.3 nM) to cells synchronized in early G1 in the absence of T3 shortened the G1 period, estimated from graphic data, from more than 40-50 h to 13.4 +/- 2.1 h (n = 7). To determine the mechanism of this T3 effect, GC cells were grown in Dulbecco's modified Eagle's medium containing 10% serum plus or minus T3 (0.3 nM) and synchronized at the beginning of the G1 period by mitotic selection. Mitotic cells (85-100%), obtained by controlled mechanical shaking, were isolated by centrifugation and replated. The end of G1 was determined by the onset of DNA synthesis with [3H]thymidine as assessed by autoradiography (percent labeled nuclei). L-T3-induced shortening of G1 was detectable at 0.05 nM T3, half-maximal at physiological T3 (0.17 nM), and maximal between 0.3 nM and 1.0 nM T3. Addition of cycloheximide, 0.025 microgram/ml or 1.0 microgram/ml, decreased protein synthesis by 50% and 90%, respectively, and attenuated the T3 effect on G1 by 80-90%. The attenuation of the T3 effect on G1 by cycloheximide at a dose which inhibited protein synthesis suggests that T3-induced shortening of G1 may require new protein synthesis. Since glucocorticoids decrease the effect of T3 on induction of alpha-aminoisobutyric acid transport, their effect on T3-induced shortening of G1 was determined in G1-synchronized GC cells and in asynchronous cultures. Cortisol, 100 nM, significantly decreased the growth rate of asynchronous GC cells and attenuated the effect of T3 in G1-synchronized cells. Finally, T4 also decreased the length of G1 in a dose-dependent manner with a half-maximal effect at 40.0 nM. The half-maximal effect of T4 occurred at a nuclear iodothyronine concentration that was comparable to that achieved in incubations with 0.17 nM T3 (half-maximal dose). Thus, half-maximal shortening of G1 in synchronized GC cell cultures occurred at iodothyronine concentrations required for half-maximal occupancy of nuclear T3 receptors and for half-maximal induction of GH synthesis, growth rate, alpha-aminoisobutyric acid uptake, and depletion of the nuclear T3 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha-Aminoisobutyric acid transport in rat soleus muscle during endotoxic shock.

We studied alpha-aminoisobutyric acid (AIB) transport by skeletal muscle and the effect of insulin and Na+ on the transport process during endotoxic shock. Rats (140-160 g) were injected with Salmonella enteritidis endotoxin (20 mg/kg iv) or saline and killed 5 h later. At that time an elevation of blood lactate and hypoglycemia marked the onset of shock in rats given endotoxin. AIB uptake was measured in isolated soleus muscles in vitro. Total cellular AIB uptake in the endotoxic muscles was 25, 28, and 47% lower than control muscles at 1-, 2-, or 3-h incubations, respectively. Insulin stimulated AIB uptake to a lesser extent in endotoxic muscles (from a basal value of 11.62 +/- 0.29 nmol X g dry wt-1 X 3 h-1 to 15.88 +/- 0.64, 19.10 +/- 1.06, and 18.78 +/- 0.52 at 1, 10, and 100 mU/ml insulin, respectively) than in controls (from 17.07 +/- 0.51 to 27.13 +/- 1.16, 27.25 +/- 0.93, and 29.01 +/- 1.09). Na+-dependent AIB uptake, calculated as the difference between AIB uptake in the presence and absence of Na+, was decreased in the endotoxic muscles to 36% of the control value. Na+-independent AIB uptake (measured in Na+-free media) was the same in control and endotoxic muscles. These results suggest that the decrease in both basal and insulin-stimulated AIB transport was due to the decrease in Na+-dependent AIB transport by skeletal muscle during endotoxic shock.

The effect of graded hypertonic intracarotid infusions on drug delivery to experimental RG-2 gliomas.

The RG-2 brain tumor model was used to determine whether unidirectional transport of alpha-aminoisobutyric acid (AIB) into brain and tumor tissue was increased after an intracarotid infusion of one of six different hypertonic solutions of L-arabinose. Intracarotid infusion of hypertonic solutions that have been reported as subthreshold for normal brain were used to determine whether they would selectively increase blood-to-tissue transport in brain tumors. No increase in the transport rate constant (K) across RG-2 tumor capillaries resulted from the infusion of 0.8- to 1.4-osm solutions. Infusions of 1.6- and 1.8-osm solutions were also performed, and blood-to-tissue transport was measured under conditions that produce maximum blood-brain-barrier disruption; however, no increase in the transport rate across tumor capillaries was measured. In brain regions surrounding the tumor, there was a trend toward increasing K values associated with increasing osmolality of the infusate, but the magnitude of this increase was small. There was a progressive increase in the K of tumor-free brain regions. This increase correlated with increasing osmolality of the infusate (0.8 to 1.8 osm). We conclude that intracarotid infusion of hypertonic solutions of L-arabinose does not increase the rate of delivery of water-soluble drugs to experimental RG-2 brain tumors. In this situation, the use of hypertonic infusions may be counterproductive and result in a greater delivery to and exposure of surrounding and normal brain tissue to levels of chemotherapeutic drugs which are potentially neurotoxic.

Characteristics of alpha-aminoisobutyric acid transport in rat skeletal muscles.

alpha-Aminoisobutyric acid (AIB) transport into the intracellular compartment of extensor digitorum longus and soleus muscles was measured (in vitro) after allowance for the equilibration of the amino acid in the extracellular space. The latter was determined with three markers, [14C]inulin, 60Co-EDTA and [3H]mannitol. Net transport of AIB was subsequently divided into its two components, i.e. influx and efflux. Rates of influx were measured as the intracellular accumulation of [14C]AIB after a short incubation (5 min), and efflux was measured as the release of AIB with time (up to maximum of 50 min) from muscles that had previously been preloaded with AIB. This intracellular efflux was resolved into two phases, which probably represent two separate components of exit. The influence of extracellular Na+ on the transport of this neutral amino acid (representing the A system) was investigated. Na+ depletion resulted in lower accumulations of AIB, the effects becoming more pronounced with progressive depletions of external Na+. These changes arose from an inhibition of AIB influx, concomitant with an enhancement of its efflux. In contrast, all components of tyrosine transport (representing the L system) were unaffected by lowering external Na+ concentrations. The net accumulation of AIB was also suppressed by cortisol. This inhibitory effect was, however, Na+-dependent and resulted solely from the steroid's enhancement of AIB efflux, the hormone being without effect on AIB influx.