Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Characterization of neutral amino acid transport in a marine pseudomonad.

The transport of neutral amino acids in marine pseudomonad B-16 (ATCC 19855) has been investigated. From patterns of competitive inhibition, mutant analysis, and kinetic data, two active transport systems with overlapping substrate specificities were distinguished and characterized. One system (DAG) served glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid (AIB) and, to a lesser extent, L-alanine and possibly other related neutral D- and L-amino acids. The other system (LIV) showed high stereospecificity for neutral amino acids with the L configuration and served primarily to transport L-leucine, L-isoleucine, L-valine, and L-alanine. This system exhibited low affinity for alpha-aminoisobutyric acid. Neither system was able to recognize structural analogues with modified alpha-amino or alpha-carboxyl groups. The kinetic parameters for L-alanine transport by the DAG and LIV systems were determined with appropriate mutants defective in either system. For L-alanine, Kt values of 4.6 X 10(-5) and 1.9 X 10(-4) M and Vmax values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for transport via the DAG and LIV systems respectively. alpha-Aminoisobutyric acid transport heterogeneity was also resolved with the mutants, and Kt values of 2.8 X 10(-5) and 1.4 X 10(-3) M AIB were obtained for transport via the DAG and LIV systems, respectively. Both systems required Na+ for activity (0.3 M Na+ optimal) and in this regard are distinguished from systems of similar substrate specificity reported in nonmarine bacteria.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

Thyroid hormone. Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have examined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5 X 10(-8) M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1 X 10(-7) M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5 X 10(-9) M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K'd1 = (0.82 +/- 0.36) X 10(-10) M and K'd2 = (3.2 +/- 0.60) X 10(-8) M. The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1 X 10(-8) M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5 X 10(-8) M T3 showed a K'd1 = (0.15 +/- 0.10) X 10(-10) M and K'd2 = (3.5 +/- 0.10) X 10(-8) M. We conclude that T3 inhibits the action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line

In the present study we investigated the characteristics of the transport of inorganic phosphate (Pi) in an opossum kidney cell line endowed with parathyroid hormone (PTH) receptors. In confluent epithelial cell culture, a Na-dependent Pi transport (NaPiT) was identified. Preincubation for 1 h with bovine (b)PTH(1-34) at 10(-7) M inhibited the NaPiT from 2.76 +/- 0.11 to 1.08 +/- 0.10 nmol/mg protein X 2 min-1 (p less than 0.001). This inhibition was already expressed 5 min after exposure to 10(-7) M bPTH. It was associated with a 4-fold increase in cellular cyclic AMP. The NaPiT was significantly inhibited at 10(-9) M bPTH, a hormonal concentration which stimulated the cellular cyclic AMP by only 30%. Kinetic analysis of the NaPiT inhibition by 10(-7) M bPTH revealed a decrease in Vmax (from 4.14 +/- 0.32 to 2.41 +/- 0.14 nmol/mg protein X 2 min-1) with no change in Km (0.093 +/- 0.016 versus 0.094 +/- 0.012 mM). The effect of bPTH on NaPiT was not associated with a change in the Na-dependent glucose methylglucopyranoside transport also present in the opossum kidney cell line. The inhibitory influence of bPTH on NaPiT was not affected by blockage of new protein synthesis by cycloheximide. Stimulation of cyclic AMP production by 10(-5) M forskolin, 10 micrograms/ml cholera toxin, 10(-5) M prostaglandin E2 or addition of 10(-5) M dibutyryl cyclic AMP mimicked the PTH-induced reduction in NaPiT. In conclusion, the present study indicates that the opossum epithelial cell line is endowed with a Na-dependent Pi transport system which is selectively inhibited by PTH and agents which increase cyclic AMP production.     

Effects of chloropyramine, dimethindene and diphenhydramine on transepithelial ion transport: studies in bovine tracheal epithelium and frog skin

The effects on transepithelial ion transports of chloropyramine, dimetindene and diphenhydramine, which are three antagonists of H1-receptors of histamine, were examined in bovine tracheal epithelium and in frog skin. The short-circuit current I0 across bovine tracheal epithelium is the sum of active secretion of Cl- and absorption of Na+. In this tissue, all three drugs induced a reversible, dose-related inhibition of I0, up to 100%. The concentrations giving 50% of maximal effect were 1.4 X 10(-4) M for chloropyramine, 2.0 X 10(-4) M for dimetindene and 2.5 X 10(-4) M for diphenhydramine. The effect was unrelated to the agonist binding site of H1-receptors of histamine, since it was not altered in the presence of 10(-3) M histamine. Experiments in which Na+ transport was selectively reduced by 5 X 10(-5) M amiloride, or in which Cl- transport was selectively abolished by 10(-3) M furosemide, 10(-4) M bumetanide or Cl- removal, indicated that Na+ and Cl- transports were equally affected by the drugs. The action of chloropyramine was composed of an early inhibition of Na+ and Cl- movements, followed by a slow recovery of Cl- secretion. In frog skin, each one of the three H1-antagonists modified the I0, following two main patterns of response, a stimulation at the lower concentrations tested, or an inhibition at higher concentrations. Dose-response relationships were obscured by a large variability in response of individual skins. These observations in bovine tracheal epithelium and frog skin suggest that H1-antagonists might alter the functioning of other epithelia as well.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles. Modulation by agents which elevate cyclic AMP

ATP-dependent Ca2+ transport was studied in basolateral membrane vesicles prepared from rat parotid gland slices incubated without or with agents which increase cyclic AMP. Isoproterenol (10(-5) M), forskolin (2 X 10(-6) M) and 8-bromocyclic AMP (2 X 10(-3) M) all increased ATP-dependent 45Ca2+ uptake 1.5- to 3-fold. The effect of isoproterenol was concentration-dependent and blocked by the beta-adrenergic antagonist propranolol. Enhanced uptake did not appear an artifact of vesicle preparation as apparent vesicle sidedness, 45Ca2+ efflux rates, specific activity of marker enzymes and equilibrium Ca2+ content were identical in vesicle preparations from control and 8-bromocyclic AMP-treated slices. Kinetic studies showed the ATP-dependent Ca2+ transport system in vesicles from 8-bromocyclic AMP-treated slices displayed a approximately 50% increase in Vmax and in Km Ca2+, compared to controls. The data suggest that physiological secretory stimuli to rat parotid acinar cells, which involve cyclic AMP, result in a readjustment of the basolateral membrane ATP-dependent Ca2+ pump.     

Myo-inositol transport through the blood-brain barrier

The unidirectional transport of [³H]myo-inositol across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured using an in situ rat brain perfusion technique. Myo-inositol was transported across the blood-brain barrier by a low capacity, saturable system with a one-half saturation concentration of 0.1 mM. The permeability surface-area product was 6.2×10^–5S^–1 with a myo-inositol concentration of 0.02 mM in the perfusate. The myo-inositol stereoisomer scyllo-inositol but not (+)-chiro-inositol (both 1 mM) inhibited myo-inositol transfer through the blood-brain barrier. These observations provide evidence that myo-inositol is transferred through the blood-brain barrier by simple diffusion and a stereospecific, saturable transport system.     

Calmodulin-induced activation of ATP-dependent Ca2+ transport in plasma membranes of the myometrium

Calmodulin activates the ATP-dependent transport of Ca2+. The V0 value for this reaction in the absence of calmodulin is 0.82, that in the presence of 10(-7) M calmodulin is 5 times as high, i. e. 4.5 nmol 45Ca2+/mg protein/min. The Vmax value in the absence of calmodulin is 2.07, that with the activator is 4.33 nmol 45Ca2+/mg protein/min. The corresponding Km values are 0.75 X 10(-6) M and 0.66 X 10(-7) M, respectively, i. e., the affinity of the Ca-pump for Ca2+ increases. The half-maximum Ca-binding activity of calmodulin measured with a help of the fluorescent probe, N-phenyl-1-naphthylamine (PNA), is observed at 5 X 10(-7) M Ca2+. Mg2+ (3 mM) decreases 10-fold the Ca-binding affinity. No significant effect of ATP on the Ca-binding properties of calmodulin was found; the Hill coefficient is suggestive of a positive cooperativity of this reaction. A comparison of dependences of the calmodulin-stimulated component of ATP-dependent transport of Ca2+ in myometrium plasma membranes and of the Ca-binding activity of calmodulin measured with a help of PNA suggests that the effect of calmodulin on the affinity of the Ca-pump for Ca2+ can also be realized when some (but not all) Ca-binding sites in the calmodulin molecule are saturated with Ca2+.     

Uptake of adrenaline by rat brain synaptosomes. Effects of several psychotropic drugs]

Comparative study of the uptake of 3H-epinephrine (3H-EN) and 3H-norepinephrine (3H-NE) into rat brain crude synaptosomes and effect of psychotropic drugs of different classes on this process showed that isolated nerve terminals had their own transport system for EN. The crude synaptosomal fraction had two transport system's for EN; high-specific active uptake with high affinity (KM = 3.7 + 0.21 microM) and low-affinity uptake (KM2 = 98.0 + 47.5 microM). En accumulation was saturable, stereo-specific and inhibited by ouabain (3 X 10(-3) M), protoveratrine A and B (10(-4) M), NaN3 (2 X 10(-3) M), 2,4-dinitrophenol (2 X 10(-3) M), p-chloromercuribenzoate (10(-4) M). Actinomycin D had no effect on the uptake of 3H-EN. 3H-HE was accumulated by two uptake system: 1-high affinity uptake system with KM values of 0.49 + 0.13 microM, 2-low affinity uptake system with KM values of 21.1 + 7.71 microM. Amphetamine, mesocarb, chlorpromazine, fluphenazine and haloperidol were equally effective inhibitors of 3H-EN and 2H-HE uptake. Imipramine, phenazepam, diazepam and carbamazepine (5 X 10(-5) M) had no effect on the uptake of 3H-NE. Imipramine, zimelidine, norzimelidine and viloxazine (5 X 10(-5) M) were more potent inhibitors of the 3H-EN uptake than that of 3H-NE.     

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 X 10(-6) M) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 X 10(-8) M for dexamethasone and 5 X 10(-7) M for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 microU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 X 10(-6) M) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy-D-glucose transport at physiological insulin concentrations, 250 microU/ml (10 ng/ml). Hydrocortisone also enhanced specific [125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.     

Cadmium stimulation of Na-independent organic acid transport in the kidney tubules

The influence of Cd++ (as well as of Hg++ and Cu++) on the uptake of an organic acid (fluorescein) in superficial proximal tubules of the surviving rat kidney was studied at 20 degrees C, when the active transport of fluorescein does not depend on the external Na. In contrast to mercury and copper, cadmium stimulated the uptake of fluorescein from the beginning of incubation. The minimal effective concentration of Cd++ was 5 X 10(-6)M, the relative effect of Cd++ on the uptake being the same within the concentration range from 5 X 10(-6) to 10(-3) M. A 60 minutes pre-incubation with Cd++ at 20 degrees C resulted in a significant increase in the stimulatory effect of acetate on the fluorescein transport. The stimulation of the fluorescein transport by cadmium was prevented by ouabain or by omissing Na from the incubating medium, although neither ouabain nor the absence of Na affected the transport of fluorescein under these conditions. It is supposed that the stimulation by Cd++ of the fluorescein transport may result from the activated oxidation of NAD-linked substrates due to acceleration of the active transepithelial transport of Na ions.     

Glucose dehydrogenase (hexose 6-phosphate dehydrogenase) and the microsomal electron transport system. Evidence supporting their possible functional relationship.

The ability of a microsomal enzyme, glucose dehydrogenase (hexose 6-phosphate dehydrogenease) to supply NADPH to the microsomal electron transport system, was investigated. Microsomes could perform oxidative demethylation of aminopyrine using microsomal glucose dehydrogenase in situ as an NADPH generator. This demethylation reaction had apparent Km values of 2.61 X 10(-5) M for NADP+, 4.93 X 10(-5) m for glucose 6-phosphate, and 2.14 X 10(-4) m for 2-deoxyglucose 6-phosphate, a synthetic substrate for glucose dehydrogenase. Phenobarbital treatment enhanced this demethylation activity more markedly than glucose dehydrogenase activity itself. Latent activity of glucose dehydrogenase in intact microsomes could be detected by using inhibitors of microsomal electron transport, i.e. carbon monoxide and p-chloromercuribenzoate (PCMB), and under anaerobic conditions. These observations indicate that in microsomes the NADPH generated by glucose dehydrogenase is immediately oxidized by NADPH-cytochrome c reductase, and that glucose dehydrogenase may be functioning to supply NADPH.     

Effect of insulin to decrease glucose transport in dissociated cells from the R3230AC mammary adenocarcinoma of diabetic rats.

Dissociated cells of the R3230AC mammary tumor were found to take up glucose by diffusion and by a passive carrier system. Using labeled 3-O-methylglucose as the probe, the following properties of the passive carrier were identified: (1) specificity for glucose, (2) competition by galactose and mannose but not by mannitol and fructose, (3) inhibition by phloretin but not by phloridzin, (4) temperature sensitivity, and (5) a Km for transport of 3-4 mM. The effects of insulin in vitro on carrier-mediated glucose transport were investigated in tumor cells from diabetic rats. At 10-9 M insulin, a time-related decrease in v for transport was observed resulting in an increased calculated Km (2- to 3-fold increase after 60-90 min incubation with insulin); only slight effects on V were obtained. This unusual response in v to insulin was observed when glucose was present in the medium at 2 mM and 5 mM, but not at 20 mM glucose. The effect of insulin to decrease the v was dose-related, with the major effects seen between 10-10M and 10-8M. The apparent decrease in glucose entry in vitro may in part explain the ability of insulin to inhibit growth of this tumor in vivo.     

Enhancement and inhibition of benzo[a]pyrene-induced SOS function in E. coli by synthetic antioxidants

8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo[a]pyrene-induced SOS function. None of the antioxidants leads to increased beta-galactosidase activity by itself. Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo[a]pyrene-induced SOS function at benzo[a]pyrene concentrations between 10(-6) M and 3 X 10(-5) M. Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo[a]pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene. Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo[a]pyrene action. In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo[a]pyrene. Preliminary data suggest that the apparent suppression of benzo[a]pyrene-induced SOS function is not due to an effect on the formation of benzo[a]pyrene metabolites by the metabolizing system used.     

Phosphate transport in fertilized sea urchin eggs. II. Effects of metabolic inhibitors and studies on differentiation

Metabolic inhibitors were applied after the transport system was fully developed in concentrations sufficient to block cleavage. 0.5–1.0 × 10^?4 M cyanide and anaerobiosis caused from negligible to moderate (40%) inhibition of phosphate uptake. The inhibition occurred late in the breeding season, and the inhibitory action of cyanide on uptake was associated with irreversible developmental effects. Azide (3 × 10^?3 M) did not inhibit uptake when the chamber method was used, but the aliquot and Hopkins' tube methods gave considerable inhibition. Purified preparations of 2,4-dinitrophenol (1 × 10^?4 M) did not inhibit uptake. Sodium iodoacetate (up to 0.05 M) and phlorizin (0.005 M) exerted no effect. Calculations of the minimal work requirement for the transport process reveal that this amounts to only a small fraction (0.24% at an external phosphate concentration of 2 μM) of the total available metabolic energy. Exposure of eggs at five minutes after insemination (lag phase) to cyanide (5 × 10^?5 M), anaerobic conditions, or azide (3 × 10^?3 M) blocked the expected increase of phosphate uptake. Removal of the inhibitors led to resumption of development and the appearance of the phosphate transport system in an essentially normal pattern. Exposure of eggs to 1.4–2.0 × 10^?4 M p-chloromercuribenzoate (p-CMB) during the accumulation phase severely depressed phosphate uptake, but cleavage was not inhibited nor delayed; recovery from the inhibition was accelerated by 1 × 10^?3 M cysteine. Exposure to p-CMB during the lag phase blocked the appearance of the transport system; cleavage proceeded normally. After the removal of p-CMB little reversal occurred until the addtion of 1 × 10^?3 M cysteine, when the phosphate transport system developed in an essentially normal manner. Trypsin (0.001–0.01%) neither activates the transport system in unfertilized eggs, nor inactivates it in denuded fertilized eggs by removal of surface proteins. The data are consistent with the conclusion that (1) the phosphate transport system is newly synthesized at fertilization in energy dependent reactions, and (2) phosphate transport is a carrier mediated process not directly dependent on metabolic energy.     

Thyroid hormone receptors in human cultured fibroblasts: evidence for cellular T4 transport and nuclear T3 binding

In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4.     

Chemical reactivity of the functional groups of insulin. Concentration-dependence studies.

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.     

Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin-induced inhibition of sugar transport.

Experiments were performed to investigate whether the fluid transported across the small intestine is isoosmotic with the mucosal solution when the active transport of glucose is partially inhibited. Everted hamster mid small intestine was incubated in one of the following four mucosal solutions: (1) Isotonic control, Krebs-Ringer bicarbonate solution containing 10 mM glucose (KRBSG), (2) Isotonic with phlorizin, KRBSG + 5X10-5 M phlorizin, (3) Hypertonic control, KRBSG + 50 mM mannitol, (4) Hypertonic with phlorizin, KRBSG + 50 MM mannitol + 5x10-5 M phlorizin. The serosal surface of the intestine was not bathed. Results indicate that the transported fluid was always isoosmotic with any of the mucosal solutions used. When the mucosal solution was made hypertonic with mannitol, the concentration of glucose and electrolytes in the absorbate increased, and as a result, the absorbate became hypertonic and isoosmotic with the mucosal solution. The presence of phlorizin either in the isotonic or in the hypertonic mucosal solution decreased the glucose concentration of the absorbate, but the transported fluid became isoosmotic with the mucosal solution due to a higher concentration of Na, K, and their associated anions. Phlorizin caused a decrease in the transmural potential difference. In spite of this, the presence of this glucoside in the mucosal solution increased the transport of sodium in relation to glucose transport. It is suggested that, at the concentrations used, phlorizin inhibits sodium movement through the electrogenic pathway, but increases the transport of this ion through the non-electrogenic route. This increase in neutral sodium transport seems to compensate for the low concentration of glucose in the absorbate, so that the absorbate becomes isoosmotic with the mucosal solution whether the latter is isotonic or hypertonic. It is suggested further that isoosmotic transport of fluid is an inherent property of the small intestine and that there may be an osmoregulatory mechanism in the gut which controls this process.     

Connective tissue activation: stimulation of glucose transport by connective tissue activating peptide III

Connective tissue activating peptide III (CTAP III), a human platelet derived growth factor, induced marked stimulation of 2-deoxy[14C]glucose (2dG) uptake in cultures of human synovial cells, chondrocytes, and dermal fibroblasts. Cytochalasin B (2 X 10(-5) M) blocked the mediator-induced increase in 2dG uptake; phlorhizin (8 X 10(-4) M) partially inhibited this process. When cells were exposed to CTAP III (4 X 10(-6) M) for 30 min prior to uptake assay, 2dG uptake was stimulated by 30-110%; greater stimulation (400-800%) occurred following 17-40-h preincubation with the mediator. A 17-h exposure to CTAP III similarly stimulated 3-O-methylglucose uptake by over 400%, suggesting that CTAP III stimulated 2dG uptake is mediated via changes in hexose transport. Cycloheximide clearly prevented the 17-h effects of CTAP III on 2dG uptake. Insulin (3 X 10(-6) M) stimulated 2dG uptake 40-70% after 30-min preincubation with hormone; little effect was seen after 17-h preincubation. These data suggest that CTAP III stimulates glucose transport shortly after addition to target cells; the major stimulation observed after a 17-h incubation is consistent with the synthesis of new glucose transport protein.