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Biomedical subjects

P Washko

Publications and source records attributed to P Washko.

7 recordsLinked to original sources
PubMed1992

Inhibition of ascorbic acid transport in human neutrophils by glucose.

Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure.

Adult↗
PubMed1991

Ascorbic acid in human neutrophils.

The uptake and distribution of ascorbic acid and the effect of extracellular glucose on ascorbic acid transport were investigated in human neutrophils. Freshly isolated neutrophils contained 1.0-1.4 mmol ascorbic acid/L, at least 94% of which was present unbound in the cytosol. Intracellular ascorbic acid was found only in the reduced form. The presence of physiologic amounts of ascorbic acid in the extracellular buffer led to the accumulation of millimolar concentrations of ascorbic acid intracellularly. Accumulation was mediated by a high- and a low-affinity transport activity. The high-affinity transport activity had an apparent Km of 2-5 mumol/L whereas the low-affinity transport activity had an apparent Km of 6-7 mmol/L. Glucose inhibited the uptake and accumulation of ascorbic acid by both transport activities in a concentration-dependent fashion. Glucose-induced inhibition of both ascorbic acid transport activities was completely reversible.

Ascorbic Acid↗
PubMed1990

Ascorbic acid accumulation in plated human neutrophils.

Ascorbic acid uptake was investigated in isolated, plated human neutrophils using high-performance liquid chromatography with coulometric electrochemical detection. Freshly isolated neutrophils contained 1.3 mM ascorbic acid and accumulated significantly greater amounts when physiologic concentrations of the vitamin were present in the extracellular buffer. In several different buffers uptake was dependent on the presence of calcium and magnesium. Under these conditions, scintillation spectrometry of [14C]ascorbic acid in conjunction with high-performance liquid chromatography was suited for measuring ascorbic acid transport.

Ascorbic Acid↗
PubMed1989

Ascorbic acid transport and accumulation in human neutrophils.

The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake.

Ascorbic Acid↗
PubMed1989

Ascorbic acid within chromaffin granules. In situ kinetics of norepinephrine biosynthesis.

Ascorbic acid requirements for norepinephrine biosynthesis were investigated in intact bovine chromaffin granules using the physiologic substrate dopamine and a novel coulometric electrochemical detection high pressure liquid chromatography system for ascorbic acid. 10 mM external dopamine, 1 mM Mg-ATP, and 1 mM ascorbic acid produced maximal norepinephrine biosynthesis without granule lysis. When external ascorbic acid was omitted, intragranular ascorbic acid was consumed in a 1:1 ratio with respect to norepinephrine biosynthesis. The initial concentration of intragranular ascorbic acid was 10.5 mM, which was depleted in stepwise fashion to 15 lower concentrations over the range of 9.2-0.2 mM. Chromaffin granules containing these varying concentrations of intragranular ascorbic acid were then incubated with 1 mM exogenous ascorbic acid, and norepinephrine biosynthesis from dopamine was determined. The apparent Km of norepinephrine biosynthesis for intragranular ascorbic acid was 0.57 mM by Eadie-Hofstee analysis and 0.68 mM by Lineweaver-Burk analysis. These data indicate that intragranular ascorbic acid is available and required for norepinephrine biosynthesis, that ascorbic acid is a true co-substrate for dopamine beta-monooxygenase, and that intragranular ascorbic acid is maintained by extragranular ascorbic acid. Continued norepinephrine biosynthesis in granules is dependent on both intragranular and extragranular concentrations of the vitamin. Furthermore, in situ kinetics of dopamine beta-monooxygenase for ascorbic acid may be most accurately determined using intact granules and the true physiologic substrate.

Adrenal Medulla↗
PubMed1977

Role of dietary calcium and calcium binding protein in cadmium toxicity in rats.

Growing male rats were fed a purified diet containing 0.6% Ca (two groups) or 0.1% Ca (two groups) for 8 weeks. One 0.6% Ca group and one 0.1% Ca group received 25 ppm Cd (as CdC12) in the drinking water. Diets were fed on an equalized basis with the 0.1% Ca + Cd group determining the amount of diet fed to the other groups. Water was provided ad libitum. Terminal body weights were not different among the four groups. Packed cell volumes were depressed in the Cd-exposed groups, especially the 0.1% Ca + Cd group. The highest concentrations of Cd were found in the lungs, liver, and kidneys of the 0.1% Ca + Cd group. More Cd was bound to low molecular weight proteins of the intestinal mucosa from the 0.1% Ca + Cd group than the 0.6% Ca + Cd group. Rats fed the 0.1% Ca diet appeared to have a greater capacity to absorb either Ca or Cd than rats fed the 0.6% Ca diet, as shown by an enhanced binding of 45Ca and 115mCd to intestinal calcium-binding protein (CaBP) in the rats fed the low calcium diet. A portion of the mucosal Cd was accounted for as Cd bound to metallothionein. It was concluded, based upon these experiments, that cadmium retention and signs of toxicity are enhanced by feeding low Ca diet and that the increased CaBP activity due to Ca restrictions is responsible for the increased Cd uptake observed.

Animals↗