I. Bihler

Transport of Nitrogen Mustard on the Transport-Carrier for Choline in L5178Y Lymphoblasts

The uptake of choline by L5178Y lymphoblasts occurs by a carrier mechanism and is an active process. Transport of nitrogen mustard and its hydrolyzed derivative is mediated by this same carrier. This finding is an example of drug transport by a carrier whose primary function is transport of a naturally occurring substrate.

Sugar transport at the basal and lateral aspect of the small intestinal cell

Abstract A method has been developed for the study of sugar transport across the basal and lateral aspect of epithelial cells from mouse small intestine. Suspensions of siolated epithelial cells were prepared from everted intestine which had been treated with HgCl 2 , an irreversible inhibitor of active sugar transport. Since sugar transport at the luminal aspect was blocked, transport at the other faces could be studied in isolation. This transport process is independent of Na + and insensitive to phlorizin. Its specificity differs from that of the Na + -dependent active process.

The action of cardiotonic steroids on sugar transport in muscle, in vitro☆

Abstract 1. (1) The membrane transport of sugar in the “intact” rat hemidiaphragm in vitro was studied by measuring the distribution of 3-O- methyl- d -[ 14 C]glucose into the intracellular water. Transport was significantly increased in the presence of ouabain and several other cardiac glycosides and aglycones at concentrations (about 10−5 M) known to inhibit Na+ transport. 2. (2) This effect required preincubation of the tissue with the drug and 10 mM glucose and was enhanced by submaximal concentrations of insulin (0.5 munit/ml) in the incubation or preincubation medium. It would seem that a transport and/or binding step is a prerequisite for the action of these drug. 3. (3) Stimulation of transport by ouabain was inhibited by phlorizin or N-ethylmaleimide and was absent when transport was fully activated by supramaximal concentrations of insulin, indicating that cardiotonic steroids affect the stereospecific, insulin-sensitive sugar transport process. 4. (4) Stimulation of transport was prevented by anoxia or uncoupling of oxidative phosphorylation, showing that the process affected by the drug depends on respiratory ATP. 5. (5) Incubation of a K+-free medium produced a stimulatory effect identical in every manner with that of ouabain. This suggests that the effect of cardiotonic steroids on sugar transport is due to their inhibition of a sodium pump. 6. (6) It is suggested there exists in muscle a negative feedback from an aerobic sodium pump to sugar transport and that this is part of the regulatory mechanism whereby the Pasteur effect is exerted at the level of membrane transport.

Intestinal sugar transport: Ionic activation and chemical specificity

The mode of absorption of several monosaccharides was studied in vitro with segments and everted sacs of hamster small intestine. 1. 1. Na+ reduced the apparent Km of transport of the actively transported d-galactose, 3-methyl-d-glucose, α-methyl-d-glucoside, d-xylose and l-glucose. The ‘nontransported’ d-arabinose, l-arabinose, l-rhamnose, l-mannose and l-fucose showed Michaelis-Menten kinetics, but their Kms were not significantly altered by Na+. The νmax of all the sugars tested was identical and independent of Na+. Inhibition of entry of nontransported sugars by d-galactose and by phlorizin was demonstrated. 2. 2. By the criteria of Na+ dependence, inhibition by phlorizin and ouabain and inhibition by transported sugars, d-mannose and d-fructose were also shown to interact with the joint sugar carrier. The transport of d-mannose appears to be active, but rapid metabolism of the two sugars precluded a quantitative determination of transport parameters. 3. 3. The data suggest a dual specificity of a joint sugar carrier: In the absence of Na+ its affinity for many diverse sugars is low; the presence of Na+ increases the affinity for only some of these sugars. The potential for active transport depends on the extent of this activation by Na+ and varies with different sugars from negligible to very great. 4. 4. With low concentrations of sugar the asymmetry of transport increases towards a limiting value which depends on the ratio Km(Na+free)Km(Na+). Active transport occurs with sugars where this ratio >3−5, provided their concentration is below a critical value which also parallels the Km ratio.

The effect of lithium on intestinal sugar transport

Abstract 1. 1.The sugar uptake of hamster small intestine in vitro was measured with various replacements for Na + in the medium. None of them supported transport against a concentration difference, but sugar equilibration was significantly more rapid in the presence of Li + than of mannitol, choline or K + . 2. 2.This stimulatory effect of Li + was inhibited by 0.5 mM phlorizin and was observed with 3-O- methyl- d -glucose , d -galactose and α-methyl- d -glucoside, which are all actively transported in the presence of Na + , but not with l -fucose and l -arabinose. Mutual inhibition between 3-O- methyl- d -glucose and d -galactose was observed in Li + medium but not in mannitol medium. 3. 3.Li + had no effect on the entry of α-aminoisobutyric acid. 4. 4.It is suggested that Li + stimulates sugar entry by activating the sugar “carrier” and not by interacting with the Na + pump. These findings are consistent with the hypothesis of Crane et al. 3,4 that intestinal sugar active transport is mediated by an ion-activated carrier.

The association of lysophosphatidylcholine with isolated cardiac myocytes

The ability of exogenous lysophosphatidylcholine to produce electrophysiological derangements and cardiac arrhythmias in the heart has been documented. The action of lysophosphatidylcholine is thought to be mediated via its association with the membrane. The present study examined the nature of the association of lysophosphatidylcholine with isolated rat myocyte membrane. The association was studied by incubating myocytes in a lysophosphatidylcholine-containing medium. The association of lysophosphatidylcholine with the myocyte sarcolemma was not affected by palmitic acid and glycerophosphocholine but was reduced by platelet-activating factor (PAF). The addition of albumin (5 mg/mL) at the end of the incubation period effectively removed the lysophosphatidylcholine from the myocytes. Our results suggest that most of the lysophosphatidylcholine in isolated myocytes was associated preferentially with the outer leaflet of the myocyte sarcolemma. This type of association might be responsible for the lysophosphatidylcholine-induced electrophysiological alterations in the heart.

Effects of diphenylhydantoin on the transport of Na+ and K+ and the regulation of sugar transport in muscle in vitro☆

Abstract 1. 1. The distribution of the nonmetabolized glucose analog 3-O- methyl- d -[ 14 C]-glucose and the intracellular concentration of Na+ and K+ were measured in “intact” rat hemidiaphragms, in vitro. 2. 2. 5,5-Diphenylhydantoin (DPH) at concentrations of 0.1 and 0.5 mM produced an increase in internal K+ and a decrease in internal Na+, consistent with stimulation of the Na+ pump. Higher concentrations, 1.0 and 5.0 mM had the opposite effect. Both the stimulatory and the inhibitory effects were potentiated by increased external K+ (16 mM). 3. 3. Sugar transport was inhibited by DPH whenever internal K+ was increased and Na+ decreased and, conversely, sugar transport was stimulated following the opposite ionic changes. The changes in sugar transport were highly and significantly correlated to changes in internal Na+ and K+. These effects were apparent on efflux, as well as on influx of sugar. Inhibition was present with and without insulin but was greater in the presence of the hormone. The inhibitory effect of DPH was abolished by 10−5 M ouabain, which blocks the Na+ pump but not by 10−9 M ouabain. 4. 4. The ionic changes are evidence for a stimulatory effect of DPH on ion transport which changes to inhibition at higher drug concentrations. The effects on sugar transport provide additional evidence for inhibition of sugar transport by low internal Na+ (or high K+) and support the postulated regulatory effect of internal Na+ and/or K+ on the sugar transport mechanism in skeletal muscle.

Characteristics of the membrane transport of sugars in the lens of the eye

1. 1. Membrane transport of the nonmetabolized glucose analog 3-O-methyl-d-glucose was measured in vitro in the lens of the eye of the rat. 2. 2. Transport leads to complete equilibration of the sugar and conforms to Michaelis-Menten kinetics, with a very high Km, around 90 mM. These results, together with the chemical specificity established earlier, are consistent with the operation of a facilitated diffusion process. There was no significant contribution of a parallel diffusional pathway. 3. 3. The specific blocker of sugar transport phloretin acts in the lens as a non-competitive inhibitor with a Ki of about 0.6 mM. 4. 4. Various agents and treatment known to stimulate sugar transport in muscle and adipose tissue were ineffective in thel ens. These include insulin (also when given in vivo), proteolytic enzymes, decreased extracellular Na+, changes in intracellular Na+ and K+ (inhibition of the Na+ pump), inhibition of energy metabolism by uncouplers of oxidative phosphorylation or inhibitors of glycolysis, and adrenaline. Thus, sugar transport in the lens resembles that in the mature mammalian erythrocyte. 5. 5. These data support the generalization that metabolic and hormonal modulation of sugar transport is present only in tissues where transport is rate-limiting and where glucose utilization varies in response to functional activity.

Regulation of sugar transport in muscle: Effect of increased external potassium in vitro☆

Abstract 1. (1) Membrane transport of 3-O- methyl- d [ 14 C]glucose and intracellular levels of Na+ and K+ were measured in “intact” rat hemidiaphragms, in vitro. 2. (2) Incubation in media with high K+ content led to decreased Na+ and increased K+ levels in the muscle cells, corresponding to stimulation of the Na+ pump. 3. (3) In parallel with the decrease in internal Na+ and increase in internal K+ levels, sugar transport both into and out of the cells was decreased. This effect was greater in the presence of a submaximal concentration of insulin but was also observed in its absence. 4. (4) “High-potassium” media were prepared by isosmotic substitution of K+ for Na+ or by addition of KCl to normal medium. The resulting decrease in external Na+ or increase in osmolarity, respectively, both stimulated sugar transport and partially counteracted the inhibitory effect of high K+. 5. (5) The inhibition of sugar transport by high K+ is the converse to the previously described stimulation of sugar transport following inhibition of the Na+ pump. The results thus support our earlier suggestion that the internal concentrations of Na+ and/or K+ may regulate sugar transport in muscle.

The role of calcium in stimulation of sugar transport in muscle by lithium

We have investigated the relation between the stimulation of sugar transport by Li+ and Li+-induced changes in cellular Ca2+ distribution. The fluxes of 3-O-[14C]methyl-D-glucose and 45Ca were measured in hemidiaphragm, soleus, and cardiac muscles of the rat, and cellular levels of Ca2+, Na+ and K+ were determined. Li+ increased in parallel the fluxes of 3-O-[14C]methyl-D-glucose and 45Ca in rat hemidiaphragm and soleus muscles. Sugar transport and Ca2+ efflux were also stimulated by Li+ in Ca2+-free medium, suggesting that in addition to increasing sarcolemmal Ca2+ influx, Li+ may also cause the release of Ca2+ from intracellular storage sites, presumably the mitochondria. Mitochondria were isolated from preparations of rat ventricular muscle exposed to Li+, and their Ca2+ content was determined. In rat cardiac muscle, Li+ stimulation of sugar transport was associated with decreased mitochondrial Ca2+ levels (indicating mitochondrial Ca2+ release) only under conditions of deteriorating mitochondrial function. Thus, Li+-induced changes in cellular Ca2+ distribution, which would increase cytosolic Ca2+ levels, were associated with stimulation of sugar transport. These observations support the hypothesis that the increased availability of cytosolic Ca2+ regulates the activity of the sugar transport system in muscle.