Mary Jean C. Holland

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin febroblast cultures

Abstract The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [14C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [14C]adenine or [14C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.

Etorphine pharmacokinetics in the rat: Experimental data and mathematical model

Rats were injected i.v. with 3H-etorphine (200 ng/Kg). At least 85% of the dose was cleared from the blood in the first 2 min. Blood levels continued to fall slowly from about 5% of the administered dose after 15 min to less than 2% after 3 hours. Although more than 15% of the dose was found in the liver and kidney after 15 min, labeled material did not further accumulate in these organs, but decreased to about 3% after 3 hours. The concentration of labeled material (dpm/mg tissue) in cerebellum was less than half that attained in other brain regions at early time points, probably reflecting the low number of opiate receptors in this region. After 2 hours, however, there was little difference between cerebellum and other brain regions. The highest brain concentrations observed were at the earliest time point examined (7 min). An open four compartment kinetic model was constructed to fit the data for etorphine concentrations in (i) plasma, (ii) cerebellum, and (iii) brain (excluding cerebellum). The model has 3 spatial compartments: plasma, brain, and all tissues other than brain. Etorphine in brain occupies either of 2 functional compartments: one representing receptor-bound ligand and the other, the sum of free and nonspecifically bound ligand. The dissociation rate constant for etorphine in vivo obtained by fitting model equations to data was 0.06 min-1, similar to that obtained in vitro in the presence of 150 mM sodium ion.

Transport of hypoxanthine by human diploid skin fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase.

Summary Transport of purine bases and nucleosides by a variety of mammalian cell lines is generally accomplished by facilitated diffusion, a non-concentrative, saturable process. However, previous investigators have been unable to detect a saturable component for the transport of hypoxanthine by human fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase, implying that in normal cells the enzyme actively participates in transport. In the present study we have used the phenomenon of countertransport to demonstrate the existence of a saturable transport mechanism in hypoxanthine-guanine phosphoribosyltransferase-deficient human diploid skin fibroblasts.

Inhibition by Levorphanol and Related Drugs of Amino Acid Transport by Isolated Membrane Vesicles from Escherichia coli

Levorphanol inhibits the transport of the amino acids proline and lysine by cytoplasmic membrane vesicles derived from Escherichia coli. The degree of inhibition increases with increasing levorphanol concentration and ranges from 26% at 10−6 M levorphanol to 92% at 10−3 M levorphanol. The effect is independent of the energy source, since levorphanol inhibits proline uptake to the same extent in the presence of 20 mM d-lactate or 20 mM succinate and in the absence of an exogenous energy source. Levorphanol does not irreversibly alter the ability of membrane vesicles to transport proline, since incubation of membrane vesicles for 15 min in the presence of 0.25 mM levorphanol, a concentration which inhibits proline transport by more than 75%, has no effect on the rate of proline transport by these vesicles once the drug is removed. Both the maximum velocity and the Km of proline transport are modified by levorphanol, hence, the type of inhibition produced by levorphanol is mixed. The inhibitor constant (Ki) for levorphanol inhibition of proline transport is approximately 3 × 10−4 M. Membrane vesicles incubated in the presence of levorphanol accumulate much less proline at the steady state than do control vesicles. Furthermore, the addition of levorphanol to membrane vesicles preloaded to the steady state with proline produces a marked net efflux of proline. Levorphanol does not block either temperature-induced efflux or exchange of external proline with [14C]proline present in the intravesicular pool. Dextrorphan, the enantiomorph of levorphanol, and levallorphan, the N-allyl analogue of levorphanol, inhibit proline and lysine transport in a similar manner. Possible mechanisms of the effects of these drugs on cell membranes are discussed.

Effect of Morphine Analogues on Chemotaxis in Escherichia coli

Pretreatment of Escherichia coli w3110 with levorphanol, a morphine analogue, reduced chemotaxis to serine, aspartic acid and galactose. This decreased chemotaxis was not due to decreased viability or motility. Pretreatment with 1.1 mM-levorphanol for 1 h, followed by washing to remove the drug prior to determination of chemotaxis, inhibited chemotaxis to each of the attractants by at least 80%. Pretreatment with dextrorphan, the enantiomorph of levorphanol, or levallorphan, the N-allyl analogue of levorphanol, resulted in a similar inhibition of chemotaxis. Reversal of the inhibition produced by pretreatment with levorphanol required a period of growth of at least one generation time.