(Na---K)-stimulated adenosinetriphosphatase in isolated intestinal villus tip and crypt cells

The migration of intestinal epithelial cells from the crypt area to the villus tip is associated with progressive differentiation of these cells. The distribution of (Na⁺---K⁺) stimulated adenosinetriphosphatase ((Na⁺---K⁺)-ATPase; EC 3.6.1.3) along the intestinal villus may have functional as well as developmental implications. To define this distribution, rat jejunal and ileal segments were incubated in vitro with a citrate solution that dissociates epithelial cells sequentially from villus tip to crypt area. ATPase activity in cell collections from villus tips and crypt areas were compared. The specific activity of (Na⁺---K⁺)-ATPase was higher in the villus tip than in the crypt cells of both jejunum and ileum. Crypt cell (Na⁺---K⁺)-ATPase activity in the jejunum and ileum were similar. Thus, (Na⁺---K⁺)-ATPase activity of villus tip cells in the jejunum was greater than in the ileum. There was no difference in villus tip and crypt cell Mg²⁺-ATPase activity in either jejunum or ileum. The steep gradient for (Na⁺---K⁺)-ATPase along the intestinal villus may signify an improtant difference in Na⁺ transport between the villus tip and crypt area. The higher level of (Na⁺---K⁺)-ATPase activity in the jejunal villi is consistent with the more important role of the jejunum in Na⁺ and substrate-linked Na⁺ transport.     

Age-related changes in the distribution and frequency of myeloid and T cell populations in the small intestine of calves

Mucosal dendritic cells (DCs) play a key role in discriminating between dietary antigens, commensal microflora and pathogens but little is known regarding age-related changes in mucosal DC populations. We analyzed lymphoid and myeloid populations within the epithelium and lamina propria (LP) of the ileum and jejunum of weaned calves (6 months old) and compared their frequency and distribution with newborn calves (3–5 weeks old). CD4, CD8 and γδ TcR T cells and CD11c^HiMHC Class II⁺ myeloid cell frequency were significantly different when comparing ileum and jejunum of weaned calves. In particular, the number of CD8 and γδ TcR T cells, and CD11c^HiCD14⁺ macrophages was significantly greater in the ileum but CD11c⁺ and CD11b⁺ myeloid cell distribution was similar throughout the mucosal epithelium of the small intestine. Furthermore, significant age-related changes were apparent when comparing the frequency and abundance of mucosal leukocyte subpopulations in newborn and weaned calves. Total mucosal leukocytes (CD45⁺) increased significantly with age in both ileum and jejunum and much of this increase was attributed to mucosal T cells (CD3⁺). In particular, CD4 T cells and NK cells increased significantly in the jejunum and CD8, and γδ TcR T cells increased significantly with age throughout the small intestine. In contrast, CD11c^HiMHC Class II⁺ myeloid cells remained numerically unchanged with age but DCs (CD13⁺, CD26⁺, CD205⁺) were enriched and macrophages (CD14⁺, CD172a⁺) were depleted in older animals. Therefore, regional differences between ileal and jejunal mucosal leukocytes changed with age and there was also a marked age-dependent change in the composition of mucosal myeloid cells. These observations have significant implications for host responses to both pathogens and commensal microflora.     

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig,sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na⁺ gradient (Na _out ⁺ >Na _in ⁺ ). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na⁺ gradient. Unlike selenate uptake, selenite uptake was not Na⁺ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of⁷⁵Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na⁺-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na⁺-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na⁺ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of⁷⁵Se from⁷⁵Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.     

Transport of selenate and selenite across the brush border membrane of rat and sheep small intestine

Mucosal uptake of⁷⁵Se-labeled selenate and selenite across the brush border was investigated in sheep and rat small intestine, using 3-min mucosal exposures. Uptake of selenate and selenite occurred faster in rat than in sheep small intestine. With the exception of sheep duodenum, mucosal selenate uptake was Na⁺-dependent in sheep and rat small intestine. Mucosal uptake of selenite across the brush border was Na⁺-dependent only in sheep midjejunum, whereas it was Na⁺-independent in sheep duodenum and ileum and the rat whole small intestine. Various anions inhibited selenate transport in the presence of Na⁺ in sheep midjejunum in the order S₂O₂ ^2- = CrO₄ ^2- > MoO₄ ^2- and in rat ileum in the order CrO₄ ^2- = S₂O₃ ^2- > SC₄ ^2- > MoO₄ ^2-. Thiosulfate also inhibited mucosal selenite uptake in the presence of Na⁺ in sheep midjejunum. Preincubation of rat ileum with glutathione (GSH) enhanced mucosal selenite uptake, whereas selenate uptake remained unaffected. These results indicate that selenate transport across the brush border membrane is energized in part by the Na⁺-gradient. Moreover, the Na⁺-dependent transport mechanism for the Se salts apparently has an affinity for other anions (S₂O₃ ^2-, SO₄ ^2-, CrO₄ ^2-, MOo₄ ^2-). The findings further indicate that intracellular GSH plays a role in the absorption of selenite, probably by an increase of intracellular selenite metabolism. The Na⁺-independent mucosal uptake of selenate and selenite probably represents diffusion.     

Interrelationship of glutathione–cystine transhydrogenase and glutathione reductase in developing rat intestine

1. Glutathione reductase and glutathione-cystine transhydrogenase activity in supernatant fractions of whole homogenates and homogenates of mucosal and muscular layers were determined in developing rat intestine after determination of the optimum conditions for assay of the two enzymes. In jejunum from adult rat, the K(m) values for GSSG reductase and GSH-cystine transhydrogenase activities were 0.25mm-GSSG and 0.23mm-cystine respectively. 2. The two activities could be differentiated by stability studies since GSSG reductase was stable at 60 degrees C for 10min and could be stored at 4 degrees C for 24h without loss of activity. GSH-cystine transhydrogenase, on the other hand, was denatured at 60 degrees C and completely inactive after 24h storage at 4 degrees C. 3. Based on calculations of total activities, both enzymes increased from the eighteenth day until the animals were young adults. 4. Total GSSG reductase activity increased at a greater rate with age than total GSH-cystine transhydrogenase activity as evidenced by activity ratios for GSH-cystine transhydrogenase/GSSG reductase of 0.44 and 0.12 in ileum from suckling and adult rats respectively, and 0.31 and 0.24 in jejunum from suckling and adult rats respectively. 5. In mucosa from adult rats GSSG reductase was more active in the ileum than in the jejunum, whereas GSH-cystine transhydrogenase activity was higher in the jejunum. 6. GSH-cystine transhydrogenase was active only in the muscle cells of the ileum of 7-day-old rats but became localized primarily in the mucosal layer in the adult rat. However, GSSG reductase activity was distributed evenly between the two layers throughout the intestine.     

Mitochondrial UCP4 attenuates MPP+- and dopamine-induced oxidative stress,mitochondrial depolarization,and ATP deficiency in neurons and is interlinked with UCP2 expression

Mitochondrial uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis. We explored the neuroprotective role of UCP4 with its stable overexpression in SH-SY5Y cells, after exposure to either MPP⁺ or dopamine to induce ATP deficiency and oxidative stress. Cells overexpressing UCP4 proliferated faster in normal cultures and after exposure to MPP⁺ and dopamine. Differentiated UCP4-overexpressing cells survived better when exposed to MPP⁺ with decreased LDH release. Contrary to the mild uncoupling hypothesis, UCP4 overexpression resulted in increased absolute ATP levels (with ADP/ATP ratios similar to those of controls under normal conditions and ADP supplementation) associated with increased respiration rate. Under MPP⁺ toxicity, UCP4 overexpression preserved ATP levels and mitochondrial membrane potential (MMP) and reduced oxidative stress; the preserved ATP level was not due to increased glycolysis. Under MPP⁺ toxicity, the induction of UCP2 expression in vector controls was absent in UCP4-overexpressing cells, suggesting that UCP4 may compensate for UCP2 expression. UCP4 function does not seem to adhere to the mild uncoupling hypothesis in its neuroprotective mechanisms under oxidative stress and ATP deficiency. UCP4 overexpression increases cell survival by inducing oxidative phosphorylation, preserving ATP synthesis and MMP, and reducing oxidative stress.     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

Enzymatic degradation of luteinizing hormone releasing hormone (LHRH) by mucosal homogenates from the intestine of the common brushtail possum (Trichosurus vulpecula)

The peptidolytic activity of fresh and frozen mucosal homogenates from five regions (duodenum, jejunum, ileum, caecum and colon) of possum intestine from Trichosurus vulpecula towards human Luteinizing Hormone Releasing Hormone (LHRH) was investigated. The rank of order of specific peptidolytic activity of the mucosal homogenates was jejunum > ileum > caecum> duodenum = colon, with a 3 to 4 fold difference between the least and the most active segment in both frozen and fresh samples. The formation of peptides LHRH (1-3), LHRH (1-4) and LHRH (1-5) suggest endopepetidase-24.18, endopeptidase-24.15 and angiotensin converting enzyme (ACE) might be responsible for the peptide degradation in mucosal homogenates. The inhibition of LHRH degradation by mucosal homogenates was evaluated in four regions (jejunum, ileum, caecum and colon) of possum intestine. Ethylenediaminetetraacetic acid (EDTA, 5 mM), sodium deoxycholate (SDA, 10 mM) and bacitracin (3.5 or 9 mM) inhibited the degradation of LHRH in mucosal homogenates from small intestine and hindgut. However, the serine protease inhibitor, soybean trypsin-chymotrypsin inhibitor (SBTI), did not prevent degradation of LHRH. It is concluded that combining peptides with inhibitors may enhance oral delivery of bioactive peptides or proteins to possums.     

Ontogeny of d-Mannose Transport and Metabolism in Rat Small Intestine

Oral mannose therapy is used to treat congenital disorders of glycosylation caused by a deficiency in phosphomannose isomerase. The segmental distribution and ontogenic regulation of d-mannose transport, phosphomannose isomerase, and phosphomannose mutase is investigated in the small intestine of fetuses, newborn, suckling, 1-month-old, and adult rats. The small intestine transports d-mannose by both Na⁺-dependent and Na⁺-independent transport mechanisms. The activities of both systems normalized to intestinal weight peak at birth and thereafter they decreased. In all the ages tested, the activity of the Na⁺-independent mechanism was higher than that of the Na⁺/mannose transport system. At birth, the Na⁺-independent d-mannose transport in the ileum was significantly higher than that in jejunum. Phosphomannose isomerase activity and mRNA levels increased at 1 month, and the values in the ileum were lower than in jejunum. Phosphomannose mutase activity in jejunum increased during the early stages of life, and it decreased at 1 month old, as does the amount of mannose incorporated into glycoproteins, whereas in the ileum, they were not affected by age. The phosphomannose isomerase/phosphomannose mutase activity ratio decreased at birth and during the suckling period, and increased at 1 month old. In conclusion, intestinal d-mannose transport activity and metabolism were affected by ontogeny and intestinal segment.     

Ketone-body utilization by homogenates of adult rat brain

The regulation of ketone-body metabolism and the quantitative importance of ketone bodies as lipid precursors in adult rat brain has been studied in vitro. Utilization of ketone bodies and of pyruvate by homogenates of adult rat brain was measured and the distribution of¹⁴C from [3-¹⁴C]ketone bodies among the metabolic products was analysed. The rate of ketone-body utilization was maximal in the presence of added Krebs-cycle intermediates and uncouplers of oxidative phosphorylation. The consumption of acetoacetate was faster than that ofd-3-hydroxybutyrate, whereas, pyruvate produced twice as much acetyl-CoA as acetoacetate under optimal conditions. Millimolar concentrations of ATP in the presence of uncoupler lowered the consumption of ketone bodies but not of pyruvate. Indirect evidence is presented suggesting that ATP interferes specifically with the mitochondrial uptake of ketone bodies. Interconversion of ketone bodies and the accumulation of acid-soluble intermediates (mainly citrate and glutamate) accounted for the major part of ketone-body utilization, whereas only a small part was oxidized to CO₂. Ketone bodies were not incorporated into lipids or protein. We conclude that adult rat-brain homogenates use ketone bodies exclusively for oxidative purposes.     

On the amount of (Na+ + K+)-ATPase available for transepithelial sodium ion transport in the amphibian skin

Pretreatment of frog skin epithelium homogenates with sodium dodecyl sulphate in the presence of ATP reveals levels of ouabain-sensitive ATPase activity usually higher and occasionally far higher than those required to sustain maximum rates of Na⁺ transport. This supports the view that Na⁺ transport involves only a fraction of the epithelial cells.     

The effects of oligomycin on energy metabolism and cation transport in slices of rat liver Inhibition of oxidative phosphorylation as the primary action

1. In slices of rat liver, oligomycin inhibited the net transport of Na⁺ and K⁺ by a maximum of 30% and endogenous respiration by 25%. These effects were not increased by a number of modifications in the incubation conditions.2. Mitochondria isolated from the slices after incubation showed respiratory control ratios that were somewhat less than in mitochondria from fresh liver, but state 3 respiration retained normal sensitivity to oligomycin.3. Low concentrations of oligomycin or cyanide reduced respiration and ATP levels of the slices but did not affect ion transport unless these levels fell below a definite critical value. In contrast, ouabain and atractyloside each caused substantial degrees of transport inhibition at ATP levels which were in excess of the critical value.4. High concentrations of cyanide and oligomycin reduced ATP contents maximally by 90% and 65%, respectively. Studies of lactate production, and of the effects of arsenite on respiration and ATP levels, suggested that substrate-level phosphorylation in the citric-acid cycle was the major source of the oligomycin-resistant ATP synthesis.5. The results suggest that oligomycin acts in the liver slices primarily as an inhibitor of oxidative phosphorylation, and that this is the cause of the partial inhibition of ion transport. The oligomycin-resistant ion-transporting activity is consistent with the persisting level of ATP synthesis.     

Studies on the relationship between glycolysis and (Na++K+)-ATPase in cultured cells

In several tissues a coupling between glycolysis and (Na⁺+K⁺)-ATPase has been observed. We report here studies on the coupling of glycolysis and (Na⁺+K⁺)-ATPase in Rous-transformed hamster cells and Ehrlich ascites tumor cells. The rate of (Na⁺+K⁺)-ATPase was estimated by the initial rate of ouabain-sensitive K⁺ influx after K⁺ reintroduction to K⁺-depleted cells. Experiments were performed with cells producing ATP via oxidative phosphorylation alone (i.e., lactate sole substrate), glycolysis alone (i.e., glucose as substrate in the absence of oxygen or with antimycin A), or glycolysis and oxidative phosphorylation (i.e., glucose as substrate in the presence of oxygen). The cells produced ATP at approximately the same rate under all of these conditions, but the initial rate of K⁺-influx was approx. 2-fold higher when AtP was produced from glycolysis. Changes in cell Na⁺ due to other transport processes related to glycolysis, such as Na⁺-H⁺ exchange, Na⁺-glucose cotransport, and K⁺-H⁺ exchange were ruled out as mediators of this effect on (Na⁺+K⁺)-ATPase. These data suggest that glycolysis is more effective than oxidative phosphorylation in providing ATP to (Na⁺+K⁺)-ATPase to these cultured cells.     

Energy metabolism of reticulocytes: Two different sources of energy for Na+K+-ATPase activity

Total energy production in rabbit reticulocytes amounted to 136·52 ± 6·50μmol ATP h^?1ml^?1 of reticulocytes: 88·3 per cent was provided by oxidative phosphorylation, whereas only 11·7 per cent by aerobic glycolysis. Na⁺K⁺-ATPase accounted for 23 per cent, i.e. 27·65 ± 2·55μmol ATP h^?1ml^?1 of reticulocytes, in the overall energy consumption in reticulocytes of rabbits. Under basal conditions ATP for Na⁺K⁺-ATPase activity was derived exclusively from oxidative phosphorylation. However, when the activity of Na⁺K⁺-ATPase was increased due to the stimulation of adenylate cyclase by (?)-isoprenaline, the additional energy required was provided by aerobic glycolysis. These results indicate that two different compartments, one cytosolic and the other mitochondrial, provide energy for Na⁺K⁺-ATPase activity in reticulocytes.     

Oxidative phosphorylation in Zymomonas mobilis

The obligately fermentative aerotolerant bacterium Zymomonas mobilis was shown to possess oxidative phosphorylation activity. Increased intracellular ATP levels were observed in aerated starved cell suspension in the presence of ethanol or acetaldehyde. Ethanolconsuming Z. mobilis generated a transmembrane pH gradient. ATP synthesis in starved Z. mobilis cells could be induced by external medium acidification of 3.5–4.0 pH units. Membrane vesicles of Z. mobilis coupled ATP synthesis to NADH oxidation. ATP synthesis was sensitive to the protonophoric uncoupler CCCP both in starved cells and in membrane vesicles. The H⁺-ATPase inhibitor DCCD was shown to inhibit the NADH-coupled ATP synthesis in membrane vesicles. The physiological role of oxidative phosphorylation in this obligately fermentative bacterium is discussed.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonyl cyanide m-chlorophenylhydrazone     

Flow cytometric characterization of the lymphocyte composition in a variety of mucosal tissues in healthy rhesus macaques

Background Rhesus monkeys play a central role in model studies on human infectious diseases, and often mucosal organs are affected by these pathogens, e.g. HIV. However, a comparative investigation into lymphocyte composition from different mucosal tissues is still missing. Methods Lymphocyte composition of duodenum, jejunum, ileum, colon, vagina, cervix, uterus and bronchoalveolar lavage from healthy rhesus monkeys was characterized in detail by flow cytometry. Moreover, we compared the lymphocyte proportions from intestinal biopsies with resections. Results All mucosal tissues exhibited higher values of CD8⁺, CD4⁺ CCR5⁺ and CD45RA^? memory T cells than blood, but similar levels of total T cells. Especially within the four gut sites, the lymphocyte composition varied significantly. The relative proportions of lymphocyte subsets from duodenal and colonic biopsies compared to resections differed. Conclusion The lymphocyte composition highly varies between different mucosal sites, and data obtained from biopsy and necropsy samples were mostly not comparable.     

Effect of morphine in vitro on the oxidative phosphorylation in rat liver mitochondria]

The rates of respiration in the presence of ADP and of phosphorylation as an ATP-ase activity of rat liver mitochondria was inhibited was in vitro by morphine with Ki=6.5 mM. The uncoupler-stimulated respiration of the mitochondria and the activity of ATP-ase and synthesis of ATP in the submitochondrial particles were not altered in the presence of morphine. It is suggested that morphine inhibited the adenine nucleotide transport through the mitochondrial membrane     

α-Synuclein and mitochondrial bioenergetics regulate tetrahydrobiopterin levels in a human dopaminergic model of Parkinson disease

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP⁺ toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP⁺ toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP⁺-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP⁺ exposure. We demonstrate that MPP⁺ significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.     

Role of Oxidative Metabolism in the Energy Suppply of Active Potassium Transport in Erythrocytes of the Lamprey Lampetra fluviatilis

To study role of glycolysis and oxidative metabolism in providing active transport of monovalent cations, isolated erythrocytes of the lamprey Lampetra fluviatlis were incubated at 20°C in the presence of various metabolic inhibitors. The active (ouabain-sensitive) K⁺ (⁸⁶Rb) influx into erythrocytes did not change after cell incubation for 1–2 h in the absence of glucose or in the presence of 10 mM deoxy-D-glucose or 1 mM monoiodoacetate. Inhibitors of oxidative phosphorylation (antimycin A, rotenone, sodium azide, cyanide) produced a significant decrease (on average, by 74% ) in the active K⁺ transport in the lamprey erythrocytes. All blockers of oxidative phosphorylation produced the same degree of inhibition of the K⁺ transport after the cell pre-incubation with them for 30 and 60 min. In experiments with rotenone, the K⁺ influx was reduced statistically significantly as early as in 5 min of cell incubation and reached a maximal effect after 10–20 min. The intracellular ATP content in erythrocytes decreased by 17, 37, and 45% after 5, 10, and 20 min of cell incubation with rotenone, respectively. The active K⁺ transport in the lamprey erythrocytes is most likely to be closely associated with the intracellular ATP concentration. The data obtained indicate that the energy supply of the Na,K-pump in the lamprey erythrocytes is due exclusively to oxidative phosphorylation processes.