2001 Volvo Award Winner in Basic Science Studies: Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc.

STUDY DESIGN: Disc cell viability was analyzed in relation to nutrient supply and cellular demand in vitro in a diffusion chamber. OBJECTIVE: To determine relations among nutrient supply, nutrient concentrations. and cell viability. SUMMARY OF BACKGROUND DATA: Although a fall in nutrient supply has long been thought the cause of disc degeneration in vivo, little information exists about the effects of nutrient levels or supply on cell viability and metabolism. METHODS: Isolated bovine nucleus cells were cultured in agarose gels in a diffusion chamber up to 13 days. Nutrients were supplied to the open sides of the chamber and diffused through the gel to the center, 12.5 mm away from the nutrient supply, in a configuration analogous to that of the disc in vivo. Profiles of cell viability and concentration of glycosaminoglycans across the chamber were measured in relation to cell density and medium composition. RESULTS: Cells remained viable across the chamber at low cell densities. However, at higher densities, cells in the center of the chamber died. The viable distance from the nutrient supply fell with an increase in cell density. Glucose was a critical nutrient. Survival was also poor at acidic pH (6.0). At 0% oxygen, disc cells survived up to 13 days with no loss of viability, but produced very little proteoglycan. CONCLUSIONS: The results support the idea that maximum cell density in the disc is regulated by nutritional constraints, and that a fall in nutrient supply reduces the number of viable cells in the disc and thus leads to degeneration.

Transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite Plasmodium falciparum.

The growth of the human malaria parasite, Plasmodium falciparum, within its host erythrocyte is reliant on the uptake of a number of essential nutrients from the extracellular medium. One of these is pantothenic acid, a water-soluble vitamin that is a precursor of coenzyme A. In this study we show that normal uninfected erythrocytes are impermeable to pantothenate but that the vitamin is taken up rapidly into malaria-infected cells via a transport pathway that has the characteristics (furosemide sensitivity, nonsaturability) of previously characterized, broad specificity permeation pathways induced by the intracellular parasite in the host cell membrane. The transport of pantothenate therefore constitutes a critical physiological role for these pathways. Inside the parasitized cell pantothenate undergoes phosphorylation, the first step in its conversion to coenzyme A. Parasites within saponin-permeabilized erythrocytes were shown to take up and phosphorylate pantothenate, consistent with the intracellular parasite having both a pantothenate transporter and a pantothenate kinase. Comparisons of the rate of phosphorylation of pantothenate by lysates prepared from uninfected and infected erythrocytes revealed that the pantothenate kinase activity of the P. falciparum trophozoite is some 10-fold higher than that of its host cell and that most, if not all, of the phosphorylation of pantothenate within the malaria-infected cell occurs within the intracellular parasite. These results contrast with those of previous studies in which it was proposed that the avian malaria parasite Plasmodium lophurae lacks pantothenate kinase (as well as the other enzymes for the synthesis of coenzyme A) and is reliant upon the uptake of preformed coenzyme A from the host cell cytosol.

Glucose uptake in Plasmodium falciparum-infected erythrocytes is an equilibrative not an active process.

The uptake of glucose into human erythrocytes infected with Plasmodium falciparum was investigated using a number of different glucose analogues. In short time-courses with cells suspended in media containing 5 mM glucose, 2-deoxy-D-glucose equilibrated rapidly between the intracellular and extracellular compartments. Its transport into the infected cell was primarily via the host cell (cytochalasin B-sensitive) transporter. 2-Deoxy-D-glucose did permeate the broad-specificity pathway that is induced in infected cells by the intracellular parasite. However, this pathway made little contribution to the total uptake of 2-deoxy-D-glucose under physiological conditions. In parasitised cells incubated with [14C]2-deoxy-D-glucose for prolonged periods the intracellular concentration of radiolabel increased to values higher than that in the external medium; it reached a maximum value three to six times higher than the extracellular concentration before falling back to a concentration similar to that outside the cells. This transient intracellular accumulation of radiolabel was due entirely to the phosphorylation of the [14C]2-deoxy-D-glucose and its consequent trapping within the cell. The specific characteristics of the 2-deoxy-D-glucose uptake time courses measured under different conditions were accounted for by the kinetics of the phosphorylation process and the energy status of the cell. The data indicate that 2-deoxy-D-glucose (as well as the non-phosphorylated compounds 3-O-methyl-D-glucose and L-glucose) enter the intracellular parasite via a passive (i.e. equilibrative) rather than an active (i.e. concentrative) transport process.

In search of a selective inhibitor of the induced transport of small solutes in Plasmodium falciparum-infected erythrocytes: effects of arylaminobenzoates.

Following invasion of the human erythrocyte by the malaria parasite, Plasmodium falciparum, there appear in the parasitized cell new, high-capacity permeation pathways that transport a diverse range of low-molecular-mass solutes. In this study a series of 16 arylaminobenzoates, analogues of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), were tested for their effects on the transport of choline, a univalent cation, into malaria-infected cells. A number of the arylaminobenzoates were found to be potent inhibitors of malaria-induced choline transport and to be similarly effective at blocking the induced transport of the uncharged pyrimidine nucleoside thymidine and the univalent anion lactate. The data are consistent with the hypothesis that much of the induced transport of cations, anions and non-electrolytes into parasitized cells is via broad-specificity, anion-selective pathways of a single type. A comparison of the effects of the arylaminobenzoates on malaria-induced transport with their effects on a number of representative anion transport systems in normal mammalian cells suggests that it is possible to identify pharmacological agents that block the malaria-induced pathway while not significantly affecting important transport mechanisms in host tissues. The most potent of the induced-transport inhibitors identified were shown to inhibit [3H]hypoxanthine incorporation in in vitro parasite growth assays. These data support the view that the induced-transport pathway may be a viable pharmacological target.

Novel anion dependence of induced cation transport in malaria-infected erythrocytes.

Following invasion by the malaria parasite there appear in the parasitized erythrocyte new ("induced") permeation pathways that mediate the transport of a wide variety of small solutes. Although anion-selective, these pathways have a significant cation permeability and cause a substantial increase in the basal leak of cations into and out of the infected cell. In this study of human erythrocytes infected in vitro with Plasmodium falciparum it was shown that the transport of monovalent cations (Rb+ and choline), but not that of a nonelectrolyte (sorbitol) or a monovalent anion (lactate), via the malaria-induced pathways is strongly dependent on the nature of the anion in the suspending medium. Substitution of NO3- for Cl- resulted in a 4-6-fold increase in the unidirectional influx and efflux of Rb+, and a 2-3-fold increase in the influx of choline via the induced pathways. By contrast, replacement of Cl- with NO3- caused a slight (although not significant) decrease in the malaria-induced influx of sorbitol and lactate. Hemolysis experiments with a range of K+ salts revealed that the net influx of K+ into infected cells showed the same novel anion dependence as seen for the unidirectional flux of Rb+ and choline, with hemolysis occurring much faster in iso-osmotic KNO3 and KSCN solutions than in KCl, KBr, or KI solutions. Hemolysis in the corresponding Na+ salt solutions was very much slower, consistent with the induced pathways being selective for K+ over Na+, and raising the possibility that the efflux of cell K+ via these pathways may play a role in host cell volume regulation. A number of models that would account for the anion dependence of malaria-induced cation transport are considered.

Transport of diverse substrates into malaria-infected erythrocytes via a pathway showing functional characteristics of a chloride channel.

Following infection by the malaria parasite, Plasmodium falciparum, human erythrocytes show increased permeability to a variety of low molecular weight solutes. In this study a number of anion transport blockers were identified as potent inhibitors of the transport of a wide range of solutes into human erythrocytes infected in vitro with P. falciparum. 5-Nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB), furosemide, and niflumate blocked the malaria-induced transport of monovalent cations, neutral amino acids, sugars, nucleosides, and monovalent anions. For all of the substrates tested the order of potency of these three inhibitors was the same (NPPB > furosemide > niflumate) and dose-response curves for the effect of these inhibitors on malaria-induced choline transport were similar to those for malaria-induced thymidine transport. The data suggest that much, if not all, of the high capacity (non-saturable) transport of low molecular weight solutes into P. falciparum-infected erythrocytes is via a single type of pathway. The broad specificity of the pathway, its non-saturability in the physiological concentration range, and its failure to distinguish between stereoisomers (L- and D-alanine) are consistent with its being a type of pore or channel. For those substrates for which quantitative influx measurements were made the magnitude of the malaria-induced (inhibitor-sensitive) transport was in the order: Cl- > lactate > thymidine, adenosine > carnitine > choline > K+. The pathway is therefore anion-selective. The pharmacological and substrate-selectivity properties of the pathway show marked similarities to those of chloride channels in other cell types; this raises the possibility that the high capacity transport of small organic solutes may be an important and, as yet, largely unrecognized role for such channels in other tissues.

Glibenclamide and meglitinide block the transport of low molecular weight solutes into malaria-infected erythrocytes.

Following infection by the malaria parasite, human erythrocytes show increased uptake of a wide variety of low molecular weight solutes via pathways with functional characteristics different from those of the transporters of normal erythrocytes. In this study glibenclamide and meglitinide were shown to inhibit the induced transport of a sugar alcohol (sorbitol), an amino acid (threonine), an inorganic anion (Cl-) and an organic cation (choline) into human erythrocytes infected in vitro with Plasmodium falciparum. The results are consistent with the hypothesis that a diverse range of substrates enter malaria-infected cells via common pathways which have features in common with Cl- channels in other cell types. glibenclamide and meglitinide were also shown to inhibit the in vitro growth of the intracellular parasite which would suggest that these pathways may be a viable chemotherapeutic target.

C value and cell volume: their significance in the evolution and development of amphibians.

Cell volume has been determined in 18 species of amphibian, ranging in C value from 1.4 pg to 62 pg DNA. There is a strong linear relationship between C value and both erythrocyte volume and erythrocyte nuclear volume. We have collected data on the timing of early embryogenesis from fertilization of the egg to the hatching tadpole in some amphibians ranging in C value from 1.4 pg to 83 pg. The species with large genomes take up to 24 times longer to reach a comparable state of development. Polyploid species develop faster than closely related diploid species. These data are discussed in relation to genome expansion and increase in cell cycle time as factors in the evolution of the Amphibia.