Intravesicular pH Research Articles

Characterization of a distinct Na(+)-H+ exchanger in basolateral membranes of human jejunum

Kinetically distinct Na(+)-H+ exchangers exist on the apical and basolateral membranes of rabbit ileal enterocytes. The apical Na(+)-H+ exchanger appears to function in electroneutral NaCl transport, whereas the basolateral Na(+)-H+ exchanger may function in homeostatic intravesicular pH (pHi) regulation and volume regulation. This study is designed to determine the presence and characteristics of the Na(+)-H+ exchanger in basolateral membrane vesicles (BLMV) prepared from jejunal tissues of human organ donors. A well-validated Percoll-gradient technique was used to prepare BLMV. An outwardly directed H+ gradient [pHi/extravesicular pH (pHo) = 5.2/7.5] resulted in a Na+ uptake overshoot (1.45 +/- 0.21 nmol/mg protein) 2.5-fold above equilibrium values (0.59 +/- 0.13 nmol/mg protein). Na+ uptake at equilibrium represented transport into an osmotically sensitive intravesicular space as validated by an osmolality study. Na+ uptake represented an electroneutral process, as shown by studies in which negative membrane potentials were induced by K+ and the ionophore valinomycin. Na+ uptake was linear for the first 15 s of transport as depicted by y = 0.042x + 0.002, r2 = 0.98. Dixon plot analysis of amiloride sensitivity revealed an ID50 value for amiloride of 29 microM (fourfold lower than ID50 for brush-border Na(+)-H+ exchanger). Kinetic studies of amiloride-sensitive Na+ uptake revealed a maximal velocity = 1.53 +/- 0.19 nmol.mg protein-1.5 s-1 and Michaelis constant = 9.83 +/- 3.5 mM. By varying pHi a sigmoidal effect of internal H+ on Na+ uptake was noted consistent with an internal modifier site for protons. To confirm this finding, the effect of pHi on Na+ efflux and Na(+)-Na+ exchange was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of chicken intestinal brush border membrane Ns/H exchange

1. 1. Na/H exchange is the major pathway for Na uptake in brush border membrane vesicles from chicken small intestine. Hanes-Woolf analysis demonstrated that Na and H competed at the same extravesicular site. The K Na for Na + at extravesicular pH 6.6 is 35 mM and at pH 7.4, 12 mM. 2. 2. Similar to mammalian intestinal cells, the Na/H exchanger does not appear to have an internal proton modifier site. Varying intravesicular pH from 6.1 to 7.8 stimulates uptake, but a sigmoidal relationship is not observed. 3. 3. The ability of several amiloride analogs to inhibit the exchanger was tested and the inhibitory profile was similar, but not identical to Na/H exchangers in mammalian tissues. The potency series (from most to least potent) is hexamethylamiloride ≈ ethylisopropylamiloride > methylisobutylamiloride > dimethyl-amiloride > amiloride.

Transport of methotrexate in basolateral membrane vesicles from rat liver

Transport of the antifolate cancer drug methotrexate was studied in vesicles isolated from the basolateral membrane of rat liver. Transport of methotrexate by basolateral membrane vesicles (BLMVs) was mostly via uptake into an osmotically active intravesicular space, with some binding (approximately 9%), as shown by initial uptake studies and by varying medium osmolarity with increasing concentrations of sucrose. Methotrexate transport was linear for the first 20 s of incubation. Transport was not affected by imposition of a Na + gradient across the vesicular membrane. Transport of methotrexate displayed a broad pH optimum: at an intravesicular pH of 7.5, the initial rate of uptake was not significantly different at extravesicular pH values ranging from 5.5 to 7.5, but uptake was less at extravesicular pH of 5.0 or 8.0. Methotrexate transport was saturable: K m = 0.15 ± 0.05 μM and V max = 11.4 ± 1.1 pmol 10 s −1 mg −1 protein. Methotrexate uptake into BLMVs was not inhibited by 5-methyltetrahydrofolate nor by 5-formyltetrahydrofolate but was weakly inhibited by folic acid in a concentration-dependent manner. Uptake was also inhibited by the anion-exchange inhibitor 4,4′-diisothio-cyanostilbene-2,2′-disulfonic acid (DIDS), and by the structurally unrelated anions ATP, ADP, Cl −, SO 4 2−, and oxalate 2−. Adenosine (no negative charge) had no effect on transport. When vesicles were preloaded with anions (ADP, SO 4 2−, oxalate 2−) such that an anion gradient existed from the intra- to the extravesicular compartment, and methotrexate uptake was measured, no stimulation of uptake was seen. Methotrexate uptake into rat liver BLMVs was electrogenic as shown by stimulation of the initial rate of uptake by a valinomycin-imposed K + diffusion potential across the vesicular membrane. These results suggest that methotrexate is transported into the hepatocyte across the basolateral membrane by an electrogenic, multispecific anion carrier system.

Carrier-mediated transport of tetrabromosulfonephthalein by rat liver plasma membrane vesicles.

To investigate the molecular requirements and mechanisms for the hepatic uptake of phthaleins, the transport of tetrabromosulfonephthalein (TBS) was investigated in basolateral rat liver plasma membrane vesicles. TBS uptake was electrogenic as greatly accelerated by the creation of a positive-inside membrane potential by the addition of valinomycin in the presence of an inwardly directed potassium gradient. No effect was observed when the ionophore was added in the presence of a sodium gradient. The transport occurred into an osmotic-sensitive space and was saturable with an apparent Michaelis constant of 5.32 +/- 0.56 microM and a maximal velocity of 9.23 +/- 0.25 nmol.s-1.mg protein-1 (mean +/- SD, n = 3 experiments). TBS uptake was directly related to the extra-vesicular pH, indicating the deprotonated quinoid negative-charged form of the dye as the transported species. In contrast, TBS uptake was inversely related to the intravesicular pH, suggesting that protonation inside the vesicles may act as an efficient trap in transport process. Addition of polyclonal monospecific anti-bilitranslocase antibody to liver vesicles specifically inhibited TBS uptake rate (3.27 +/- 0.17 vs. 5.82 +/- 0.61 nmol.s-1.mg protein-1, n = 3, P less than 0.001). These data indicate that TBS is electrogenically transported across the liver cell plasma membrane by bilitranslocase. They also indicate that the presence of a negative charged group on the benzenic ring of the ligand is important in accounting for the transport.

Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes.

The ADP-ribosylation factor (ARF), a highly conserved low molecular weight GTP-binding protein, has been implicated to function in intracellular protein transport to and within the Golgi complex. In pancreatic acinar cells the ARF is confined to the cytoplasmic faces of trans-Golgi stack membranes, a compartment known to maintain a low intravesicular pH, which is established by a chloride-dependent MgATP-driven proton pump. The present study shows that MgATP (2mM), but neither adenosine 5'-[gamma-thio]triphosphate in the presence of Mg2+ nor ATP in the absence of Mg2+, increases transfer of ARF from the surrounding medium into the vesicle membranes. The specific vacuolar-type proton pump inhibitor bafilomycin B1 (10 nM), the protonophore carbonylcyanide m-chlorophenylhydrazone (10 microM), and replacement of chloride in the incubation buffer by acetate or nitrate resulted in an almost complete inhibition of the MgATP-dependent association of ARF to the vesicle membranes. The results demonstrate that redistribution of ARF to the vesicle membrane correlates with the intravesicular pH established by a vacuolar-type H(+)-ATPase. The intravesicular pH appears to be one mechanism by which certain low molecular weight GTP-binding proteins become relocated from the cytosol to their specific membrane vesicles.

Ca2+ transport through the brush border membrane of human placenta syncytiotrophoblasts.

The calcium (Ca2+) uptake by brush border membrane vesicles isolated from fresh human placentas has been characterized. This process was saturable and time- and concentration-dependent. It exhibited a double Michaelis-Menten kinetics, with apparent Km values of 0.17 +/- 0.03 and 2.98 +/- 0.17 mM Ca2+, and Vmax values of 0.9 +/- 0.13 and 2.51 +/- 0.45 pmol.micrograms-1.5 s-1. It was not influenced by the presence of Na+ or Mg2+ in the incubation medium. It was not increased by K+ or anion diffusion potentials, inside negative. At a steady state of 1 mM Ca2+ uptake, a large proportion (approximately 94%) of the Ca2+ was bound to the internal surface of the membranes. Preincubation of these membrane vesicles with voltage-dependent Ca2+ channel blockers (nifedipine and verapamil) had no influence on Ca2+ uptake. However, this uptake was very sensitive to pH. In the absence of a pH gradient, the Ca2+ uptake increased with alkalinity. When the intravesicular pH was kept constant while the pH of the incubation medium was increased, Ca2+ uptake was also stimulated by alkaline pH. In contrast, when the pH of the incubation medium was kept constant and the intravesicular pH was progressively increased, Ca2+ uptake was diminished with alkaline pH. Therefore, H+ gradient (H+ in trans-position greater than H+ in cis-position) favored Ca2+ transport, suggesting a H+/Ca2+ exchange mechanism. Finally, in contrast to the basal plasma membrane, the brush border membrane did not show any ATP-dependent Ca2+ transport activity.

In vivo potentiation of ricin toxicity by monensin delivered through liposomes

Monensin, a carboxylic ionophore which is known to raise intravesicular pH, was intercalated in liposomes and its effect on the toxicity of ricin in mice was studied. The toxicity of ricin in vivo was found to be significantly enhanced by the administration of monensin intercalated in liposomes (liposomal monensin). The observed enhancement of the toxicity of ricin by monensin was highly dose-dependent and was maximal when ricin was injected within 60 min of monensin injection. The survival time was found to be reduced in the range of 8–20 h, depending on the dose of ricin used, by liposomal monensin. Stability of liposomes containing monensin as inferred from the release of entrapped calcein or FITC-dextran under both in vivo and in vitro conditions was comparable to that observed for liposomes without monensin. Liposomal monensin remains in circulation for 2 h and was cleared from the blood stream after 4 h. In contrast, 15 min was required for the clearance of monensin when administered in free form. Studies on the distribution of liposomal monensin and 125I-ricin in various tissues have revealed that monensin is mainly localized in the liver and spleen which are also the major sites for ricin accumulation. Our observation on the substantial enhancement of ricin toxicity in vivo by liposomal monensin strongly supports the potential usefulness of the latter as a potentiating agent in the enhancement of the toxicity of immunotoxin or hormonotoxin for selective elimination of cancer cells.

Association of a 19- and a 21-kDa GTP-binding protein to pancreatic microsomal vesicles is regulated by the intravesicular pH established by a vacuolar-type H(+)-ATPase.

Evidence suggests that certain ras-related small molecular weight GTP-binding proteins (smg-proteins) are involved in intracellular membrane trafficking and vesicle fusion. We have previously shown that intravesicular acidification due to a vacuolar-type H(+)-ATPase, which is Cl- dependent and highly sensitive to the specific inhibitor bafilomycin, enhances GTP-induced fusion of pancreatic microsomal vesicles (Hampe, W., Zimmermann, P., Schulz, I. 1990. FEBS Lett. 271:62-66). This process may involve function of smg-proteins. The present study shows that MgATP (2 mM), but neither MgATP gamma S nor ATP in the absence of Mg2+, increases association of 19- and 21-kDa smg-proteins to the vesicle membrane as monitored by their [ alpha-32P]GTP binding. The affinity of smg-proteins for [ alpha-32P]GTP was not altered by MgATP. Bafilomycin B1 (10(-8) M), the protonophore CCCP (10(-5) M), and replacement of Cl- in the incubation buffer by CH3COO- or NO3- resulted in an almost complete inhibition of the MgATP-dependent association of the 19- and 21-kDa smg-proteins to the vesicle membranes. Furthermore, the MgATP effect on both smg-proteins was found to be due to the intravesicular pH and not to the H+ gradient over the vesicle membrane. We conclude that association of a 19-kDa (immunologically identified as the ADP-ribosylation factor, arf) and a yet unidentified 21-kDa GTP-binding protein to vesicle membranes is regulated by the intravesicular pH established by a vacuolar-type H(+)-ATPase.

Transport of 5-methyltetrahydrofolate in basolateral membrane vesicles of rat liver

We examined 5-methyltetrahydrofolate transport across the basolateral membrane (BLM) of rat liver using isolated membrane vesicles. Uptake of 5-methyltetrahydrofolate by BLM vesicles (BLMV) was found by osmolarity and initial uptake studies to be mostly the result of transport of the substrate into an active, intravesicular space with some binding (approximately 25%) to membrane surfaces. Uptake was similar in the presence of an inwardly directed Na+ or K+ gradient, indicating that transport is not dependent on Na+. Uptake of 5-methyltetrahydrofolate was linear for the first 30 s and was more rapid when an initial pH gradient was imposed across the vesicular membrane [extravesicular pH (pHo) = 5.0, intravesicular pH (pHi) = 7.5]. Under pH gradient conditions, uptake at 3-5 min was about twofold higher than at equilibrium (60 min). The initial rate of uptake at pHo = pHi = 5.0 was more rapid than at pHo = pHi = 7.5, but in neither case was uptake above equilibrium observed. Uptake under pH gradient conditions and at pH 5.0 (no pH gradient) was saturable (apparent Michaelis constants of 0.58 and 0.36 microM, respectively; maximum uptake rates of 1.25 and 0.79 pmol.10 s-1.mg protein-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Vesicle acidification driven by a millionfold proton gradient: a model for acid influx through gastric cell membranes.

A delta pH 6 proton (internal pH 7.5, external pH 1.5) was imposed across the bilayer of egg phosphatidylcholine (PC) vesicles. The presence of the gradient was verified by the use of agents that predictably collapsed it. These agents included a detergent (Triton X-100), a pore-forming Na-H+ exchanger (nigericin), and weak acids capable of shuttling protons across the membrane (acetic and acetylsalicylic acids). The magnitude of the proton gradient and the rate of pH gradient collapse were determined by measuring pyranine fluorescence by use of an isosbestic point technique and calculating intravesicular pH from a calibration curve. Acid flux into the vesicles in the presence of chloride was measured directly by a simpler one-wavelength method. p-Xylene-bis-pyridinium bromide (DPX), a pyranine fluorescence quencher, was used to verify that the fluorescence signal originated from within the vesicles and that the observed rates of acidification were not an artifact due to pyranine leakage. Acid flux was found to be dependent on the ionic composition of the buffer. The presence of chloride ion in the external compartment caused a dramatic increase in the rate of acidification. It was demonstrated that the kinetics of acid flux into the vesicle were clearly controlled by the chloride-dependent mechanism at a relatively low chloride concentration of 2.5 mM. The model presented here is a simple and sensitive method for investigating the factors that influence acidification rates across membranes when acidification is driven by extremely large proton gradients, in the presence of chloride; conditions similar to those found in the stomach.

Characterization of essential sulfhydryl groups of rat renal Na(+)-Pi cotransporter.

Sulfhydryl (SH) groups are essential for the function of the Na(+)-Pi cotransporter of renal brush-border membrane (BBM) as determined by inhibition of Na(+)-Pi cotransport by HgCl2. Recent studies suggest that essential SH groups may be present on the cytoplasmic side of the BBM. We used various maneuvers to differentiate between external and internal SH groups on the Na(+)-Pi cotransporter in renal BBM vesicles (BBMV). The inhibitory potency of p-chloromercuriphenylsulfonic acid (PCMBS), a poorly permeable SH reagent, was about one-half that of p-chloromercuribenzoic acid (PCMB), a highly permeable reagent (half-maximal inhibitory concentrations of 625 and 350 microM, respectively). 5,5'-Dithio-bis-(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) were additive to HgCl2 for inhibition of Pi transport. The highly permeable NEM gave a more pronounced additive effect (+30%) than the less permeable DTNB (+15%). When the intravesicular pH (pHi) and extravesicular pH (pHo) were varied independently, NEM (which reacts mainly at an alkaline pH) inhibited Pi transport only at pHi = 8.5, regardless of pHo. When internal SH groups were blocked by NEM at pH 8.5, PCMB and PCMBS produced similar additive effects. The binding of 14C-labeled phosphonoformic acid was inhibited by both reagents and to the same extent. Both PCMB and PCMBS increased 32Pi efflux from BBMV. The findings are consistent with the presence of essential SH groups on the cytoplasmic side of the BBM, with possible conformational changes induced by modification of the external SH groups.

The influence of pH on cystine and dibasic amino acid transport by rat renal brushborder membrane vesicles

The uptake of cystine and lysine by rat renal brushborder membrane vesicles was examined at various intravesicular and extravesicular hydrogen ion concentrations to discern whether ionic species are determinants of specificity for the shared transport system and whether hydrogen ion gradients play a role in determining uptake values. When intravesicular and extravesicular pH are identical, the highest uptake of cystine occurred at pH 7.4, with lesser uptake at pH 6.0 and 8.3. Since cystine is electroneutral at pH 6.0 and 90% anionic at pH 8.3, it appears that neither form of the amino acid is a preferred species for transport. A similar relationship between pH and uptake occurs for lysine, which is cationic at pH below 8.5. This suggests that pH affects the functioning of the membrane carrier system independent of ionic species of the substrate. There is no apparent relationship of cystine uptake to hydrogen ion gradients across the membrane. Over the range of extravesicular pH studied, optimal cystine uptake occurred whenever the intravesicular pH was 7.4. Competitive interactions between unlabeled amino acids and labeled cystine were not affected by the extravesicular pH and, therefore, did not seem determined by the ionic species of cystine.

The Mechanism of Human Placental Urea Transport: A Study Using Placental Brush Border (Microvillous) Membrane Vesicles

To elucidate the mechanism of placental amino acid transport, the transport of amino acids into brush border membrane vesicles was investigated using brush border membrane vesicles (BBMV) separated from the human full-term placenta. 1. The transport of l-alanine and l-glutamine into the brush border membrane vesicles prepared from either early gestational placenta or full-termed placenta disclosed a typical overshooting phenomenon in the presence of H+ concentration gradient (extravesicular pH greater than intravesicular pH). 2. The H(+)-dependent overshooting transport of urea into the brush border membrane vesicles disappeared in the presence of H+ ionophore. 3. The initial velocity of H+ concentration gradient dependent uptake of urea into the brush border membrane vesicles was regulated by the saturation kinetics determined by the concentration of urea. The values of Km and Vmax, calculated as the kinetic parameters of urea transport by reciprocal plotting of the initial velocity of uptake and the concentration of urea, were 10.8 mM and 410 mumol/mg protein/10 sec, respectively. The above results indicate that a transport system which transports urea in exchange for H+ exists in human full-term placental microvillous membrane.

Evidence for daunomycin efflux from multidrug-resistant 2780 AD human ovarian carcinoma cells against a concentration gradient

Using a flow-through system human multidrug-resistant 2780 AD ovarian carcinoma cells were exposed to flowing culture medium containing the anticancer agent daunomycin (5 μM). A pulse of medium containing verapamil caused increased cellular daunomycin accumulation, resulting in a dip in the fluorescence signal from daunomycin in the effluent. After passage of this pulse we observed an efflux of more than 90% of this extra accumulated daunomycin within 10 min. This daunomycin efflux against a concentration gradient provides evidence for drug efflux from these cells being an active process. After the addition of methylamine to the medium to increase intravesicular pH, the dip in the fluorescence signal was not decreased, indicating that vesicular transport was not an important component of this efflux.

Specific structural alteration of the influenza haemagglutinin by amantadine.

Amantadine hydrochloride specifically blocks the release of virus particles from H7 influenza virus infected cells. This appears to be the direct consequence of an amantadine induced change in the haemagglutinin (HA) to its low pH conformation. The effect is indirect and mediated via interaction of the drug with the M2 protein since mutants altered in this component alone are insensitive to amantadine. The timing of drug action, some 15-20 min after synthesis, and its coincidence with proteolytic cleavage indicates that the modifications to HA occur late during transport but prior to insertion into the plasma membrane. Reversal by mM concentrations of amines and 0.1 microM monensin indicates that amantadine action causes a reduction in intravesicular pH which triggers the conformational change in HA. We conclude, therefore, that the function of M2 inhibited by amantadine is involved in counteracting the acidity of vesicular compartments of the exocytic pathway in infected cells and is important in protecting the structural integrity of the acid-sensitive glycoprotein.

Ca2(+)-induced conformational change and aggregation of chromogranin A.

Chromogranin A, the most abundant protein in bovine adrenal chromaffin granules, bound calmodulin in a Ca2(+)-dependent manner, and the calmodulin-binding property was utilized to purify chromogranin A. Chromogranin A has been described in the past as a "random-coil polypeptide" with little alpha-helix or beta-sheet conformation. However, circular dichroism measurements with pure, native chromogranin A revealed relatively high alpha-helical contents (40% at the intravesicular pH of 5.5). Fluorescence studies confirmed previous observations that chromogranin A binds Ca2+ with low affinity. Considering the high concentration of Ca2+ in the secretory vesicle, the effect of Ca2+ on the secondary structure and self-association of chromogranin A was examined. Ca2+ induced a decrease of alpha-helicity of chromogranin A from 40 to 30% at pH 5.5. In contrast, at pH 7.5 the same amount of Ca2+ increased alpha-helicity of the protein from 25 to 40%. Boiling of the adrenal extract, a commonly used purification procedure for chromogranin A, resulted in the isolation of conformationally distinct chromogranin A molecule. Unlike secretory protein-I of the parathyroid gland (Gorr, S.-V., Dean, W. L., Radley, T. L., and Cohn, D. V. (1988) Bone Mineral 4, 17-25), chromogranin A aggregated rapidly in the presence of Ca2+. The extent and rate of aggregation were highly dependent on Ca2+ concentration. Although both the rate and extent of aggregation at pH 7.5 were much lower than those at pH 5.5, aggregation of chromogranin A proceeded at both pH's. In this respect, chromogranin A differs from human chromogranin C which was shown by Gerdes et al. (Gerdes, H.-H., Rosa, P., Phillips, E., Baeuerle, P. A., Frank, R., Argos, P., and Huttner, W. B. (1989) J. Biol. Chem. 264, 12009-12015) to aggregate at pH 5.2 but not at pH 7.4.

Optical response of the indicator chlortetracycline to membrane potential

Chlortetracycline is a fluorescent, Ca 2+ indicator commonly used to monitor the internal Ca 2+ concentration of membrane vesicles and organelles. We have found that the intensity of chlortetracycline fluorescence in the presence of Ca 2+-loaded liposomes is dependent on the membrane potential of the vesicles as well as the intravesicular Ca 2+ concentration. The fluorescence of chlortetracycline was lower when an inside-negative membrane potential was placed across the liposome membrane. Since chlortetracycline diffuses across the membrane in the zwitterionic form, the distribution of chlortetracycline across the membrane should not be strongly dependent on the membrane potential. However, because the proton permeability of phospholipid vesicles is relatively high, the intravesicular proton concentration is dependent on the membrane potential. The binding of Ca 2+ to chlortetracycline is dependent on pH in the range of pH 6 to pH 8. Therefore, changes in the intravesicular pH as a result of a change in the membrane potential causes relatively large changes in the chlortetracycline fluorescence signal even when there isn't a change in the Ca 2+ concentration.

How to separate the H + and OH − permeabilities: Kinetics of intravesicular pH change after medium pH jump

The kinetics of the internal pH change after the external pH jump of finite amplitude is theoretically analyzed for the suspension system of membrane vesicles in the presence of the pH buffering agent. Under some assumptions the time course of the internal pH change is analytically solved to yield an exact solution. The solution gives us an experimental procedure by which the membrane permeabilities to H + and OH − are separately determined at any desired pH.

UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP

We have examined the coupling and charge stoichiometry for UDP-GlcNAc transport into Golgi-enriched vesicles from rat liver. In the absence of added energy sources, these Golgi vesicles concentrate UDP-GlcNAc at least 20-fold, presumably by exchange with endogenous nucleotides. Under the conditions used, extravesicular degradation of UDP-GlcNAc has been eliminated, and less than 15% of the internalized radioactivity becomes associated with endogenous macromolecules. Of the remaining intravesicular label, 85% remains unmetabolized UDP-[3H]GlcNAc, and approximately 15% is hydrolyzed to [3H]GlcNAc-1-phosphate. Efflux of accumulated UDP-[3H]GlcNAc is induced by addition of UMP, UDP, or UDP-galactose to the external medium. Permeabilization of Golgi vesicles causes a rapid and nearly complete loss of internal UDP-[3H]GlcNAc, indicating that the results reflect transport and not binding. Moreover, transport of UDP-[3H]GlcNAc into these Golgi vesicles was stimulated up to 5-fold by mechanically preloading vesicles with either UDP-GlcNAc or UMP. The response of UMP/UMP exchange and UMP/UDP-GlcNAc exchange to alterations in intravesicular and extravesicular pH suggests that UDP-GlcNAc enters the Golgi apparatus in electroneutral exchange with the dianionic form of UMP.

Insulin-Mimetic Effects of Vanadate: Possible Implications for Future Treatment of Diabetes

Vanadate ions, low-molecular-weight phosphate analogues, mimic most of the rapid actions of insulin in various cell types. When administered orally to diabetic hyperglycemic rats, vanadate reaches the circulation, mimics insulin stimulation of glucose uptake and metabolism, and leads to normoglycemic and partial anabolic states. In addition, vanadate restores tissue responsiveness to insulin and hepatic glycogen levels and activates new synthesis of key enzymes for carbohydrate metabolism. This suggests that correcting hyperglycemia is sufficient to correct the typical metabolic alterations found in streptozocin-induced diabetic rats. Several weeks of oral administration of vanadate to diabetic rats has not produced detectable liver or kidney toxicity. The mechanism by which vanadate mimics the actions of insulin is still obscure. Unlike insulin, vanadate does not seem to stimulate the autophosphorylation and endogenous tyrosine phosphorylation of insulin-receptor kinase or other intracellular proteins either directly or by virtue of its known inhibitory effect on protein phosphotyrosine phosphatase. Results from many studies support a model in which vanadate activates glucose metabolism by either utilizing an alternative (insulin-independent) cascade or bypassing the early events of the insulin-dependent cascade. Either of these possibilities is of clinical importance, because early insulin events may become defective, as a result of severe hyperinsulinemia, and may contribute to insulin resistance. Alternative pathways by which vanadate may stimulate glucose metabolism, e.g., by increasing intracellular Ca2+ levels and/or regulating intracellular and intravesicular pH, are discussed. From a clinical perspective, studies should be continued in evaluating the level of vanadate toxicity after prolonged treatment and searching for agents that potentiate its insulin mimetic actions in vitro and in vivo.