Pig reticulocytes. IV. In vitro maturation of naturally occurring reticulocytes with permeability loss to glucose

Naturally occurring reticulocytes of week old piglets were used to characterize the maturation process under in vitro conditions. When the reticulocytes were suspended in tissue culture medium fortified with metabolic substrates, nearly all cells were viable after 24 hours incubation and usually more than 85% of the initial cell population survived after an 80 hour period. In cells maintained as long as a week in incubation, an adequate level of total adenine nucleotide with a large accumulation of IMP was found. In most cases, reticulocytes lose their reticular materials within two days and assume normal erythrocyte configuration. Concomitant with the morphological change, the cell volume decreases toward normal erythrocyte size, the extent of which can be accounted for by the intracellular loss of salt and accompanying water. As in the in vivo reticulocyte maturation process, reticulocytes undergoing in vitro maturation lose their membrane permeability to glucose. These findings suggest that the process of reticulocyte maturation occurring in cell culture approaches that which naturally occurs in vivo. Thus, these cells may be used to delineate the mechanism of the loss of membrane transport of glucose which normally occurs in the adult pig cells.     

Characterization of naturally occurring auxotrophic mammalian cells.

In a previous study, several cultured cell lines were detected which are naturally occurring auxotrophs. In this investigation, the enzyme deficienceis involved are described. It is demonstrated that the Chinese hamster cell lines CHO(K1), YH21, RJK-36, and CHW-1102 are deficient in cystathionase and argininosuccinate synthetase. In addition, CHO (K1) and CHW-1102 were found to lack argininosuccinate lyase. CHW-1102 cells were also found to be unable to proliferate in medium containing branched-chain alpha-keto acids in place of the corresponding L-amino acids since CHW-1102 cells lack branched-chain aminotransferase.     

A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells.

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K+ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K+-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K+ genotype of the sheep and was not associated with either "high K+"- or "low K+"-type Na+ pump. No Ca2+-dependent change in K+ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K+ channel in the mammalian red cell and the relationship between the K+ channel and the Na+ pump are discussed.     

A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K⁺ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K⁺-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K⁺ genotype of the sheep and was not associated with either “high K⁺”-or “low K⁺”-type Na⁺ pump. No Ca²⁺-dependent change in K⁺ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K⁺ channel in the mammalian red cell and the relationship between the K⁺ channel and the Na⁺ pump are discussed.     

Studies on NAD+ permeability in intact mitochondria from rabbit reticulocytes.

Functionally intact mitochondria from rabbit reticulocytes are characterized by a low NAD+ level after the preparation (0.29 nmoles NAD+ + NADH/mg protein). They are apparently impermeable for NADH and exhibit a slow net uptake of NAD+. From the increase of O2-uptake in state 3 and the increase of NADH concentration in state 4 of respiration after the addition of NAD+ we concluded that 3--10 min are necessary for the saturation with NAD+ at 23 degrees C. 2mM NAD+ extramitochondrially are not sufficient to saturate the mitochondria with NADH and probably NAD+, too. Because of the net uptake of NAD+ we assume that reticulocyte mitochondria lose NAD+ during their preparation. If they are incubated with the physiological concentration of 300 micrometer NAD+, which was found in reticulocytes, a value of 1.9 nmoles NAD+ + NADH mg protein was calculated. At an extramitochondrial NAD+ concentration of 300 micrometer, reticulocyte mitochondria exhibit an almost maximal O2-uptake in the presence of oxaloacetate or alpha-ketoglutarate. It is concluded that the mitochondria in intact reticulocytes contain the "normal" complement of NAD+ + NADH.     

Membrane stress increases cation permeability in red cells.

The human red cell is known to increase its cation permeability when deformed by mechanical forces. Light-scattering measurements were used to quantitate the cell deformation, as ellipticity under shear. Permeability to sodium and potassium was not proportional to the cell deformation. An ellipticity of 0.75 was required to increase the permeability of the membrane to cations, and flux thereafter increased rapidly as the limits of cell extension were reached. Induction of membrane curvature by chemical agents also did not increase cation permeability. These results indicate that membrane deformation per se does not increase permeability, and that membrane tension is the effector for increased cation permeability. This may be relevant to some cation permeabilities observed by patch clamping.     

Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation

Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase la can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during ‘in vivo’ reticulocytes maturation. This new hexokinase micro-heterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.     

Glucose transport kinetics in human red blood cells

D-[14C]Glucose self exchange and unidirectional efflux from human red blood cells were studied at 20 degrees C (pH 7.2) by means of the Millipore-Swinnex filtering technique whose time resolution is greater than 1 s and the continuous flow-tube method with a time resolution of greater than 2 ms. The unidirectional efflux data were analyzed using both the method of initial rates and the integrated rate equation. Simple Michaelis-Menten kinetics apply to the results obtained under both experimental conditions. In self-exchange mode, the half-saturation constant, K1/2ex, was 10 (S.E. +/- 1) mM. In unidirectional efflux mode K1/2ue was 6.6 (S.E. +/- 0.5) mM (initial rates) or by the method of integrated rates 7.7 mM, with a range of 2.7-12.1 mM, K1/2ue increasing with an increased initial intracellular glucose concentration. Our results of K1/2ex oppose previous published values of 32 mM for self exchange (Eilam and Stein (1972) Biochim. Biophys. Acta 266, 161-173) and 25 mM for unidirectional efflux (Karlish et al. (1972) Biochim. Biophys. Acta 255, 126-132) that have been used extensively in kinetic considerations of glucose transport models. Under self-exchange conditions Jmaxex was 1.8 x 10(-10) mol cm-2s-1, and in unidirectional efflux mode Jmaxue was 8.3 x 10(-11) mol cm-2s-1 (initial rates) and 8.6 x 10(-11) mol cm-2s-1 (integrated rates). We suggest that the previous high values of Jmax and in particular K1/2 are due to the use of methods with insufficient time resolution. Our results indicate that the transport system is less asymmetric than was generally accepted, and that complicated transport models developed to account for the great difference between the determined K1/2 and J max values are redundant.     

Amine and carboxylate spin probe permeability in red cells

Summary Permeabilities for a homologous series of amine and carboxylate nitroxide spin probes were measured in human red blood cells by an electron paramagnetic resonance (EPR) method. Permeabilities determined in this study are much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. This suggests that the rate-limiting region for permeation of these nonelectrolytes is somewhere in the membrane periphery rather than in the center of the membrane. The red cell membrane does not discriminate between these probes on the basis of molecular volume, as might be predicted by a simple free-volume theory of membrane permeation.     

Metabolism of naturally occurring propenylbenzene derivatives III. Allybenzene,propenyl benzene,and related metabolic products

The acidic and neutral urinary metabolites of allylbenzene, propenylbenzene, 1′-hydroxyallylbenzene, and cinnamyl alcohol were identified and related through a common metabolic scheme. The rat metabolizes allylbenzene to 1′-hydroxyallylbenzene which can rearrange to yield cinnamyl alcohol which is further metabolized. This mechanism is proposed to account for the appearance of “propenyl type” metabolites from allylbenzene compounds. Propenylbenzene is oxidized to cinnamyl alcohol. Both 1′-hydroxyallylbenzene and cinnamyl alcohol are excreted unchanged in the neutral extract when given to rats. Allylbenzene and 1′-hydroxyallylbenzene yield basic ninhydrin-positive metabolites. Allylbenzene is first oxidized on the benzylic carbon to form 1′-hydroxyallylbenzene, which is further oxidized to form phenyl vinyl ketone, which condenses with the secondary amines piperidine and dimethylamine to form tertiary aminopropiophenones (Mannich bases). Analogous compounds, cinnamyl alcohol and propenylbenzene, do not yield Mannich base metabolites. This proposed metabolic scheme is consistent with the chemical mechanism operative in the synthesis of Mannich base from allylbenzene via chromic acid oxidation followed by amine addition.     

Induction of apoptosis in tumor cells by naturally occurring sulfur-containing compounds

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Among naturally occurring products, sulfur-containing compounds (OSCs), especially garlic compounds (GCs) and isothiocyanates (ITCs), represent two important and promising chemopreventive families because of their potent chemopreventive effects in various in vivo and in vitro models. In recent years, numerous investigations have shown that sulfur-containing compounds induce apoptosis in multiple cell lines and experimental animals. In the course of apoptosis induction by GCs and ITCs, multiple signal-transduction pathways and apoptosis intermediates are modulated. In particular, modulation of MAPKs and production of reactive oxygen species (ROS) seem to play pivotal roles in apoptosis induction by most GCs and ITCs. However, the role of P53 is still controversial. Based on present knowledge, GCs and ITCs may target not only the metabolism of carcinogens but also apoptosis signaling molecules. The effects of ITCs and GCs at multiple points of cancer development make these compounds highly promising candidates in cancer chemoprevention. However, the mechanisms of their anticancer effects are not fully understood, and further studies are required, especially to elucidate the role of cell-death receptors (the extrinsic pathway) and whether these agents induce apoptotic effects in non-tumor cells.     

A possible naturally occurring tumor promoter, teleocidin B from Streptomyces.

Dihydroteleocidin B, a derivative of teleocidin B, when painted on mouse skin, caused marked induction of ornithine decarboxylase within 4 hrs. This induction of ornithine decarboxylase was inhibited by painting the skin with 13-cis-retinoic acid one hour before dihydroteleocidin B. Dihydroteleocidin B induced cell adhesion of human promyelocytic leukemia cells (HL-60) to the surface of culture flasks, and inhibited terminal differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide. Its effective dose for these actions was comparable to that of the potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate. Teleocidin B seems to be a new type of promoter of carcinogenesis.     

Glucose turnover rate in newborn rats.

In starved newborn rats an increase in glucose turnover rate was observed 4 and 6h after birth, but a dramatic fall occurred at 16h. In suckling newborn rats, no decrease in glucose turnover rate was observed at 16h. The metabolic clearance of glucose did not change in fed or starved animals. The results are discussed in relation to metabolic adaptation to extra-uterine life.     

Viruses detected in cells explanted into tissue culture from naturally occurring malignant lymphoma in rhesus monkeys

During a 4-year period 43 cases of spontaneous lymphoma occurred in macaques at the California Primate Research Center. In an attempt to determine if there might be a common viral etiology to the outbreak, lymphoma tissues from 10 rhesus monkeys were explanted into tissue culture and examined for the presence of virus. Electron microscopic studies of the cultured lymphoma cells revealed viruses morphologically characteristics of adenovirus, reovirus, foamy virus, and herpesvirus. Cell-free filtrates from tissue cultures possessing the latter three viruses produced cytopathology in virus-free indicator cells. Virus particles typical of the explant culture were demonstrable in the infected indicator cells. Type-C RNA tumor virus particles were not observed electron microscopically in any of the lymphoma tissues or lymphoma cell cultures examined.     

Pig reticulocytes. V. Development of Rb+ influx during in vitro maturation

Influx of the K⁺ analogue Rb⁺ was measured through the ouabain-sensitive Na⁺/K⁺ pump and the ouabain-insensitive “leak” pathways in Cl^? or NO in mature red cells from adult pigs and in reticulocytes naturally occurring in 7-day-old piglets. In reticulocytes, Rb⁺ influxes by the two pathways were of about equal magnitude in Cl^? (13 and 10 mmoles/liter cells × hr) and at least 25-fold larger than in mature red cells (0.5 and 0.4 mmoles/liter cells × hr). In Na ⁺ media, a portion of the ouabain-insensitive “leak” flux of Rb⁺ was Cl^? dependent (Rb⁺Cl^? transport) as NO replacement reduced Rb⁺ influx by 90% in reticulocytes and by 40% in mature red cells. The sulfhydryl reagent N-ethylmaleimide (NEM) stimulated Rb⁺Cl^? transport about twofold in reticulocytes and up to 13-fold in mature red cells. When reticulocytes matured to erythrocytes during in vitro incubation, about 90% of both ouabain-sensitive Rb⁺ pump and ouabain-insensitive Rb⁺Cl^? influx were lost. In contrast, the NEM-stimulated Rb⁺Cl^? transport changed much less throughout this period, suggesting an entity operationally but not necessarily structrually distinct from the basal Rb⁺Cl^? transport. Although the experimental variability precluded a full assessment of significant changes in the small Na⁺/K⁺(Rb⁺) pump and Rb⁺Cl^? fluxes in mature pig red cells kept for the same time period in vitro, Rb⁺ flux changes in reticulocytes appear to be maturational in nature, reflecting parallel activity transitions of Na⁺/K⁺ pump and Cl^?-dependent K⁺ fluxes in vivo.