Pantothenate Uptake Research Articles

Heterogeneity in the effects of membrane potentials on pantothenate and glucose uptakes by rabbit renal apical membranes.

1. Previous studies using renal brush-border membrane vesicles have established that both the pantothenate and the low Km (Michaelis-Menten constant), low Vmax (maximal rate) D-glucose systems have a stoichiometry of 2 Na+: 1 organic molecule. In this study, we compared the mechanisms by which the membrane potential energizes pantothenate and D-glucose uptakes by brush-border membrane vesicles isolated from the whole cortex of rabbit kidney. 2. In the absence of Na+, varying the membrane potential from +60 to -60 mV decreased pantothenate uptake, whereas D-glucose uptake was increased in a linear manner. These results suggested the existence of a conductive pathway for pantothenate in these membranes. They also suggested that the pantothenate free carrier is electroneutral, while the glucose free carrier is negatively charged. 3. In the presence of an inwardly directed Na+ gradient, varying the membrane potential from +60 to -60 mV increased Na(+)-dependent pantothenate influx linearly. In contrast, a shift from +60 to +40 mV in the membrane potential had no influence on Na(+)-dependent D-glucose influx, whereas influx was a linear function of the membrane potential from +40 to -60 mV, indicating that there is a threshold membrane potential required for membrane potential-dependent D-glucose movement to occur. 4. Kinetic studies revealed that the effect of membrane potential on pantothenate uptake is through changes in the Km, while Vmax was unchanged. On the other hand, the membrane potential exerted its effect on D-glucose transport solely on the Vmax. 5. Finally, binding studies revealed that membrane potential, both in the presence and absence of a Na+ gradient, elicited effects on phlorizin binding qualitatively similar to those observed for D-glucose transport. 6. Implications of these findings for tubular regulation of these electrogenic secondary active transport systems are discussed.

Cloning, sequence, and expression of the pantothenate permease (panF) gene of Escherichia coli

Pantothenate permease, the product of the panF gene, catalyzes the sodium-dependent uptake of extracellular pantothenate. The panF gene was isolated from an Escherichia coli genomic DNA library and subcloned into multicopy plasmids. Increased copy number of the panF+ allele resulted in increased rates of pantothenate uptake and a significant increase in the steady-state intracellular pantothenate concentration. Despite the higher levels of pantothenate, the utilization of pantothenate for coenzyme A formation was not elevated, indicating that pantothenate kinase activity is the dominant regulator of coenzyme A biosynthesis. DNA sequencing of the panF gene revealed the presence of a single open reading frame that encoded a hydrophobic protein with a molecular weight of 51,992. Sequence analysis predicts that pantothenate permease is an integral membrane protein possessing 12 hydrophobic membrane-spanning domains connected by short hydrophilic sequences. The predicted topological profile of pantothenate permease is similar to that of other membrane carriers that catalyze cation-dependent symport.

Do Dietary Levels of Pantothenic Acid Regulate Its Intestinal Uptake in Mice?

While regulation of intestinal transporters is established for other nutrients, evidence concerning regulation of intestinal vitamin transport is scanty. Hence, we compared intestinal pantothenic acid (PA) uptake in mice fed diets with high, normal and deficient PA levels. PA uptake is distributed along the small intestine, Na(+)-dependent and saturable. Signs of PA deficiency were weight loss or reduced growth, then hair loss and exudation around the eyes, then diarrhea and hindleg paralysis and splenomegaly, and finally death. Treatment of mice with an antibiotic was found to be necessary to elicit severe signs of PA deficiency, probably because mice normally can obtain PA synthesized by intestinal bacteria. Dietary PA levels had no effect on intestinal PA uptake at 5 microM. A small increase in the Vmax of uptake, observed in late-stage deficiency, is probably too small to be physiologically significant. Comparison with published results for other water-soluble vitamins suggests that intestinal transporters may be regulated only for vitamins absorbed predominantly by carrier-mediated transport and subject to natural deficiency states.

Insulin effects on pantothenic acid uptake in isolated perfused working hearts from diabetic rats.

Pantothenic acid uptake was studied in isolated working hearts from spontaneously diabetic BB Wistar and streptozocin-induced diabetic (STZ-D) rats. If insulin treatment was stopped for a 24-h period from spontaneously diabetic rats, a significant decrease in the rate of pantothenic uptake was noted (from 147.3 +/- 5.0 to 110.8 +/- 10.6 nmol.g-1 dry wt.30 min-1). Pantothenic acid uptake rates were also reduced in 48-h STZ-D rats (118.0 +/- 6.1 nmol.g-1 dry wt.30 min-1, compared to 158.2 +/- 5.3 in control rats). The decrease in pantothenic acid uptake in all diabetic animals occurred whether hearts were perfused with 1.2 mM palmitate or 1.2 mM palmitate and 11 mM glucose. If insulin (500 microU/ml) was added to the perfusion medium of hearts from spontaneously diabetic rats perfused with palmitate and glucose, a significant increase in pantothenic acid uptake was noted (from 110.8 +/- 10.6 to 167.0 +/- 9.4 nmol.g-1 dry wt.30 min-1). Insulin had no significant effect on pantothenic acid uptake in hearts from spontaneously diabetic rats perfused with palmitate alone. In STZ-D rats, insulin added to hearts perfused with palmitate and glucose resulted in a small but significant increase in pantothenic acid uptake (from 118.0 +/- 6.1 to 130.6 +/- 4.0 nmol.g-1 dry wt.30 min-1). Insulin had no effect on pantothenic acid uptake in control hearts perfused either in the presence or absence of glucose. These data suggest that insulin, in the presence of glucose, can increase pantothenic acid uptake in diabetic rats.

Insulin effects on pantothenic acid uptake in isolated perfused working hearts from diabetic rats

Insulin effects on pantothenic acid uptake in isolated perfused working hearts from diabetic rats

Regulation of pantothenic acid transport in the heart. Involvement of a Na+-cotransport system.

Pantothenic acid transport was studied in the isolated perfused rat heart and isolated sheep cardiac sarcolemmal vesicles. In the perfused heart, pantothenic acid transport was significantly greater if hearts were perfused as working hearts rather than Langendorff hearts, but was unaffected by the perfusion substrates used (11 mM glucose or 1.2 mM palmitate). Uptake rates of pantothenic acid in working hearts are dependent on perfusate concentrations of pantothenic acid (a Vmax of 418 nmol/g dry weight/30 min and a Km for pantothenic acid of 10.7 mircoM were obtained). Reduction in perfusate Na+ concentration from 145 to 105 mM (the Na+ was replaced with 40 mM choline) resulted in a small but significant decrease in pantothenic acid uptake. At 145 mM Na+, addition of a mixture of amino acids, whose uptake is Na+-dependent, resulted in a significant decrease in pantothenic acid uptake by the heart (173 +/- 5 to 132 +/- 12 nmol/g dry weight). If an inward Na+ gradient in isolated, purified sarcolemmal vesicles, was imposed, a rapid uptake of pantothenic acid was observed. Uptake rates are markedly reduced if Na+ was replaced by equimolar concentrations of K+ or if external Na+ was reduced below 40 mM. In the presence of Na+, increasing pantothenic acid concentrations resulted in an increase in pantothenic acid uptake by the vesicles. Combined, these data demonstrate that pantothenic acid is transported across the myocardial sarcolemmal membrane by a Na+-dependent mechanism, which may be common to a number of small molecules.

Pantothenate-sodium cotransport in renal brush-border membranes.

The mechanism of pantothenate transport into rabbit renal brush-border membrane vesicles was studied. Under voltage-clamped conditions, an inward NaCl gradient induced the transient accumulation of pantothenate against its concentration gradient, indicating Na+/pantothenate cotransport. K+, Rb+, Li+, NH4+, and choline+ were ineffective in replacing Na+. Pantothenate analogs, D-glucose, and various carboxylic acids did not inhibit Na+-dependent pantothenate transport, suggesting that this system is specific for pantothenate. Kinetic analysis of the Na+-dependent pantothenate uptake revealed a single transport system which obeyed Michaelis-Menten kinetics (Km = 16 microM and Vmax = 6.7 pmol X mg-1 X 10 s-1). Imposition of an inside-negative membrane potential caused net uphill pantothenate accumulation in the presence of Na+ but absence of a Na+ gradient, indicating that Na+/pantothenate cotransport is electrogenic. The relationship between extravesicular Na+ concentration and pantothenate transport measured under voltage-clamped conditions was sigmoidal: a Hill coefficient (napp) of 2 and a [Na+]0.5 of 55 mM were calculated. It is suggested that an anionic pantothenate1- molecule is cotransported with two Na+ to give a net charge of +1. The coupling of pantothenate transport to the Na+ electrochemical gradient may provide an efficient mechanism for reabsorption of pantothenate in the kidney.

Preparation of high-specific-activity d-[3- 3H]pantothenic acid

High-specific-activity d-[3- 3H]pantothenic acid (5 Ci/mmol) was prepared from commercially available β-[3- 3H]alanine employing Escherichia coli strain DV1 ( panD2 pan F1). This strain is defective in β-alanine synthesis and pantothenate uptake, and under appropriate growth conditions converted 85 to 90% of the input β-[3- 3H]alanine to extracellular d-[3- 3H]pantothenate. The radiolabeled vitamin was purified from the medium by thin-layer chromatography followed by reverse-phase high-performance liquid chromatography. The overall yield of d-[3- 3H]pantothenic acid was 30% and radiochemical purity was >99%.

Isolation and characterization of Escherichia coli pantothenate permease (panF) mutants.

Mutants of Escherichia coli K-12 defective in the pantothenate permease (panF) were isolated and characterized. The panF mutation resulted in the complete loss of pantothenate uptake and of the ability to use extracellular vitamin for growth. The growth phenotypes of panF panD, panF panB, and panF panC double mutants showed that the cytoplasmic membrane was impermeable to external pantothenate. Analysis of the intracellular and extracellular metabolites from strain DV1 (panF panD) labeled with beta-[3-3H]alanine demonstrated that a carrier-mediated mechanism for efficient pantothenate efflux remained in the panF mutant. Genetic mapping of this nonselectable allele was facilitated by the isolation of three independent Tn10 insertions close to panF. Two- and three-factor crosses located panF at minute 72 of the E. coli chromosome and established the gene order fabE panF aroE.

Pantothenate transport in Escherichia coli.

The transport system for pantothenic acid uptake in Escherichia coli was characterized. This transport system was specific for pantothenate, had a Kt of 0.4 microM, and had a maximum velocity of 1.6 pmol/min per 10(8) cells (45 pmol/min per mg [dry weight]). Pantothenate uptake was not reduced in osmotically shocked cells or by ATP depletion with arsenate, but was reduced greater than 90% by the dissipation of the membrane electrochemical gradient with 2,4-dinitrophenol. Sodium ions stimulated pantothenate uptake (Kt, 0.8 mM) by reducing the Kt for pantothenate by an order of magnitude. Intracellular pantothenate was rapidly phosphorylated, but phosphorylation of pantothenate was not required for uptake since pantothenate was the only labeled intracellular compound concentrated by ATP-depleted, glucose-energized cells. The data were consistent with the presence of a high-affinity pantothenate permease that concentrates the vitamin by sodium cotransport.

The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture.

The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane.

Effects of diabetes and fasting on pantothenic acid metabolism in rats.

The effects of fasting and diabetes on pantothenic acid (PA) metabolism were studied in rats. Tissue levels of PA and coenzyme A (CoA) and rates of [14C]PA uptake and incorporation into tissue CoA were determined. Both fasting and diabetes resulted in accelerated rates of [14C]PA uptake, higher tissue concentrations of PA, increased incorporation of [14C]PA into CoA, and elevated tissue concentrations of CoA in the liver. The concentration of PA in liver was near the Km of pantothenate kinase for PA in control animals, and increased PA uptake may, in part, account for the increased [14C]PA incorporation into CoA though an elevation in tissue PA levels. In cardiac muscle, increased [14C]PA incorporation into CoA and increased CoA levels were associated with reduced PA uptake and reduced tissue PA levels in both fasting and diabetic animals, suggesting that CoA synthesis is not controlled by substrate availability in this tissue. Uptake of [14C]PA by skeletal muscle was also reduced in diabetic animals. These data suggest that PA uptake by tissues is under metabolic or hormonal control. Decreased uptake by muscle and increased uptake by liver may represent a mechanism for shifting large body stores of PA present in muscle to the liver in which endogenous PA concentrations are normally low. In addition, both fasting and diabetes resulted in decreased urinary PA excretion, a finding that may represent a regulatory mechanism to conserve whole-body PA under these conditions.

14C]-Pantothenate Accumulation by Isolated Adipocytes from Adult Rats of Different Ages

Accumulation of radiolabeled pantothenate by isolated adult rat adipocytes was studied in an in vitro system. Maximal uptake after 1 hour incubation occurred in media containing 0.03 mM pantothenate, about 100 picograms (pg) per adipocyte and was less than 1 pg per cell. Using such saturation conditions, uptake was energy- and temperature-dependent, with about 50% maximal accumulation occurring after 5-10 minutes incubation. Uptake was decreased by the addition of unlabeled pantothenate, but not other water-soluble vitamins. The serum level of pantothenate was decreased about 25% in animals 23-25 months old compared to those 11-13 months old, yet the ability to accumulate pantothenate at saturation conditions by adipocytes was no different. These data suggest that cellular accumulation of this vitamin is specific and that the capacity to do so under optimal conditions does not change with advancing age in this system. It is not known whether in vivo suboptimal or altered rate of cellular pantothenate uptake occurs with aging, or if it remains unchanged and lower serum levels are the result of decreased dietary intake.

The Effect of Metabolic State on Incorporation of [14C]Pantothenate into CoA in Rat Liver and Heart

The effect of metabolic or hormonal status on CoA biosynthesis was studied by comparing the rates of incorporation of [14C]-panthothenate into CoA in fasted and glucose-fed rats. Rat hearts and livers were freeze-clamped 1.5 hours after intravenous injection of [14C] pantothenate. CoA, pantothenate, and other pathway intermediates were separated by chromatography of tissue extracts on DEAE cellulose paper. Compared to the fasted rats, rats forced-fed glucose 0.5 hours before pantothenate injection 69% lower incorporation of radioactivity into CoA in liver, a 69% lower specific radioactivity of liver CoA, a 63% lower specific radioactivity of liver mitochondrial CoA, and a 44% lower incorporation of radioactivity in CoA in heart. The accumulation of labeled pathway intermediates was negligible. The cysteine content of liver was equal for the two conditions. There was no difference in level of unacylated CoASH for fasted and glucose-fed rats, suggesting that hormonal effects on degree of CoA acylation are not involved in this regulator mechanism. The specific radioactivities and concentrations of pantothenate in heart or liver were also nearly equal for the two conditions, so the regulatory mechanism does not involve hormonal effects on pantothenate uptake into tissues. Thus, the effect of glucose-feeding (and related changes of metabolite and insulin-glucagon levels) on the incorporation o[14C] pantothenate into CoA is exerted at a locus on the biosynthetic and/or degradative pathway between pantothenate and CoA.

Transport of pantothenic acid in Lactobacillus plantarum.

The characteristics of pantothenic acid (PA) transport into nonproliferating cells of Lactobacillus plantarum were studied. These characteristics included the effects of pH and temperature variations and the energy requirement for transport and binding of the vitamin. At pH 7.5 and 38 °C the intracellular vitamin was in the form of phosphopantothenic acid (P-PA) and was not exchangeable with external PA. Vitamin transport appeared separate from phosphorylation since significant free PA was observed only at elevated temperatures (e.g. 45 °C) and at low pH values. PA uptake and accumulation were energy-dependent, being inhibited completely by 10−2 M iodoacetate and the absence of glucose, exhibited pH and temperature optima of 7.5 and 38 °C, respectively, and were linear with time for at least 60 min. PA uptake exhibited substrate saturation. No free PA was observed when cells were incubated in PA concentrations above saturation levels. Apparent Km and Vmax values of 7.85 × 10−7 M and 5.35 μmol of PA/mg cells (dry weight) per minute, respectively, were determined from Lineweaver–Burk plots. Competitive inhibition of transport was observed with panthenol and pantoyl taurine. Ki values were, respectively, 3.55 × 10−5 M and 3.07 × 10−6 M. Pantoyl lactone, at concentrations which inhibited growth by 78%, had no effect on transport. The data reported suggest that PA transport is an active, mediated process and that transport may limit the phosphorylation of PA into P-PA. Inhibitor studies showed that PA transport was at least 150 times more sensitive to analogue inhibition than was growth itself, suggesting that transport of PA was not a limiting factor in the growth of L. plantarum.

Phosphopantothenic acid--the major product of pantothenic acid accumulation and a significant growth stimulatn in Lactobacillus plantarum.

Non-proliferating cells of Lactobacillus plantarum accumulated about 1.3 mug of pantothenic acid (PA) per milligram dry cells when placed in a phosphate-buffered solution containing glucose, NaCl, and PA for 21 h. Under the conditions applied for PA uptake, pH 7.5 and 38 degrees C, the accumulated PA existed almost exclusively in a bound form. Free PA was observed only occasionally and then at questionably significant levels. All bound PA was released quantitatively by enzyme hydrolysis with alkaline (intestinal) phosphatase. Chromatographic analysis coupled with differential microbiological analysis of cell extracts using Saccharomyces cerevisiae, Lactobacillus helveticus, and L. plantarum identified the accumulated PA as phosphopantothenic acid (P-PA). Assay of P-PA loaded cell extracts (devoid of free PA) by L. plantarum before and after alkaline phosphatase hydrolysis showed that the growth of this primary assay organism was stimulated by 8.3%.

Correlations between pantothenate uptake, phospholipid synthesis and pantothenate-binding protein formation in Pseudomonas fluorescens P-2.

Correlations between pantothenate uptake, phospholipid synthesis and pantothenate-binding protein formation in Pseudomonas fluorescens P-2.