We have investigated the consequences of endogenous limitations in oxygen delivery for phloem transport in Ricinus communis. In situ oxygen profiles were measured directly across stems of plants growing in air (21% [v/v] oxygen), using a microsensor with a tip diameter of approximately 30 m. Oxygen levels decreased from 21% (v/v) at the surface to 7% (v/v) in the vascular region and increased again to 15% (v/v) toward the hollow center of the stem. Phloem sap exuding from small incisions in the bark of the stem was hypoxic, and the ATP to ADP ratio (4.1) and energy charge (0.78) were also low. When 5-cm stem segments of intact plants were exposed to zero external oxygen for 90 min, oxygen levels within the phloem decreased to approximately 2% (v/v), and ATP to ADP ratio and adenylate energy charge dropped further to 1.92 and 0.68, respectively. This was accompanied by a marked decrease in the phloem sucrose (Suc) concentration and Suc transport rate, which is likely to be explained by the inhibition of retrieval processes in the phloem. Germinating seedlings were used to analyze the effect of a stepwise decrease in oxygen tension on phloem transport and energy metabolism in more detail. Within the endosperm embedding the cotyledons-next to the phloem loading sites-oxygen decreased from approximately 14% (v/v) in 6-d-old seedlings down to approximately 6% (v/v) in 10-d-old seedlings. This was paralleled by a similar decrease of oxygen inside the hypocotyl. When the endosperm was removed and cotyledons incubated in a 100 mm Suc solution with 21%, 6%, 3%, or 0.5% (v/v) oxygen for 3 h before phloem sap was analyzed, decreasing oxygen tensions led to a progressive decrease in phloem energy state, indicating a partial inhibition of respiration. The estimated ratio of NADH to NAD ϩ in the phloem exudate remained low (approximately 0.0014) when oxygen was decreased to 6% and 3% (v/v) but increased markedly (to approximately 0.008) at 0.5% (v/v) oxygen, paralleled by an increase in lactate and ethanol. Suc concentration and translocation decreased when oxygen was decreased to 3% and 0.5% (v/v). Falling oxygen led to a progressive increase in amino acids, especially of alanine, ␥-aminobutyrat, methionine, and isoleucine, a progressive decrease in the C to N ratio, and an increase in the succinate to malate ratio in the phloem. These results show that oxygen concentration is low inside the transport phloem in planta and that this results in adaptive changes in phloem metabolism and function.In contrast to animals, plants lack specialized systems for oxygen distribution. Oxygen moves by diffusion from the surrounding air (21% [v/v] oxygen) through apertures in the epidermis and intercellular air spaces within the tissue (Drew, 1997). The absence of specialized systems for oxygen delivery is not generally considered to be a problem for plant growth and metabolism because most plant organs have a relatively high surface-to-volume ratio, and their respiration rates per unit volume of tissue are usually lower than in animal...

Friedreich's ataxia (FA) is an autosomal recessive disease caused by decreased expression of the mitochondrial protein frataxin. The biological function of frataxin is unclear. The homologue of frataxin in yeast, YFH1, is required for cellular respiration and was suggested to regulate mitochondrial iron homeostasis. Patients suffering from FA exhibit decreased ATP production in skeletal muscle. We now demonstrate that overexpression of frataxin in mammalian cells causes a Ca 2؉ -induced up-regulation of tricarboxylic acid cycle flux and respiration, which, in turn, leads to an increased mitochondrial membrane potential (⌬ m) and results in an elevated cellular ATP content. Thus, frataxin appears to be a key activator of mitochondrial energy conversion and oxidative phosphorylation.

We have studied the involvement of proteolytic pathways in the regulation of the Na/P i cotransporter type II by parathyroid hormone (PTH) in opossum kidney cells. Inhibition of lysosomal degradation (by leupeptin, ammonium chloride, methylamine, chloroquine, l -methionine methyl ester) prevented the PTH-mediated degradation of the transporter, whereas inhibition of the proteasomal pathway (by lactacystin) did not. Moreover it was found ( i ) that whereas lysosomal inhibitors prevented the PTH-mediated degradation of the transporter they did not prevent the PTH-mediated inhibition of the Na/P i cotransport and ( ii ) that treating opossum kidney cells with lysosomal inhibitors led to an increased expression of the transporter without any concomitant increase in the Na/P i cotransport. Further analysis by subcellular fractionation and morphological techniques showed ( i ) that the Na/P i cotransporter is constitutively transported to and degraded within late endosomes/lysosomes and ( ii ) that PTH leads to the increased degradation of the transporter in late endosomes/lysosomes.

Parathyroid hormone (PTH) inhibits proximal tubular brush border membrane Na؉ /P i cotransport activity; this decrease in the transport activity was found to be associated with a decrease in type II Na ؉ /P i cotransporter protein content in rat brush border membranes. In the present study we investigated the PTH-dependent regulation of the type II Na ؉ /P i cotransporter in opossum kidney cells, a previously established model to study cellular mechanisms involved in the regulation of proximal tubular Na ؉ /P i cotransport. We transfected opossum kidney cells with a cDNA coding for NaP i -2 (rat renal type II Na ؉ /P i cotransporter). This allowed the study of PTH-dependent regulation of the transfected NaP i -2 and of the corresponding intrinsic cotransporter (NaP i -4). The results show (i) that the intrinsic and the transfected cotransporters are functionally (transport) and morphologically (immunofluorescence) localized at the apical membrane, (ii) that the intrinsic as well as the transfected Na ؉ /P i cotransport activities are inhibited by PTH, (iii) that PTH leads to a retrieval of both cotransporters from the apical membrane, (iv) that both cotransporters are rapidly degraded in response to PTH, and (v) that the reappearance/recovery of type II Na ؉ /P i cotransporter protein and function from PTH inhibition requires de novo protein synthesis. These results document that PTH leads to a removal of type II Na ؉ /P i cotransporters from the apical membrane and to their subsequent degradation. Renal proximal tubular P i reabsorption is acutely regulated by parathyroid hormone (PTH).1 This effect involves inhibition of the brush border membrane sodium-dependent P i transport and is characterized by a decrease in the maximal transport rate (V max ) (1, 2). Two different renal Na ϩ /P i cotransporters have been cloned, classified either as type I Na ϩ /P i cotransporter or as type II Na ϩ /P i cotransporter (3-14). Both are localized at the brush border membrane in proximal tubules. Recent data documented that physiologically and pathophysiologically altered brush border membrane Na ϩ /P i cotransport involves altered brush border expression of the type II Na ϩ /P i cotransporter (15)(16)(17).In the present study we investigated the PTH-mediated regulation of the type II Na ϩ /P i cotransporter in opossum cells (OK cells); these cells have recently been shown to contain such a cotransporter (NaP i -4; Ref. 8). The validity of the opossum kidney cell model to study proximal tubular Na ϩ /P i cotransport and its regulation has been established (18 -23). With respect to PTH-dependent control of Na ϩ /P i cotransport activity, we have reported that the recovery from the PTH-mediated inhibition of Na ϩ /P i cotransport in OK cells is dependent on de novo protein synthesis. This latter observation led to the hypothesis that PTH might lead to the retrieval and degradation of the transporter (27).The aims of the present study were 2-fold: (i) to study cellular/molecular mechanisms involved in PTH-dependent control of ...