Biochemical Characteristics of The Na-alpha-Ketoglutarate Cotransport System in Proximal Convoluted and Straight Tubules of the Rabbit Kidney / 대한신장학회잡지

PURPOSE: alpha-Ketoglutarate (alphaKG), a Krebs cycle intermediate, is extensively used in the kidney as a fuel substrate and as a counter anion for organic acid secretion. It is known to be taken up by the proximal tubule cells via the brush-border as well as basolateral membranes. We explored biochemical characteristics of the brush-border and basolateral alphaKG transport systems in pars convoluta and pars recta of the proximal tubule, respectively. METHODS: Brush-border and basolateral membrane vesicles (BBMV and BLMV) were isolated from rabbit renal outer cortex and outer medulla by Percoll gradient centrifugation. Vesicular uptake of alphaKG was determined by rapid Millipore filtration method using alpha-14[C]KG as a substrate. RESULTS: Both BBMV and BLMV showed a Na-gradient dependent uphill transport of alphaKG. The systems in both membranes were similarly inhibited by Li and activated by Na (Hill coefficient of 1.4). Kinetic analyses indicated that the Na-alphaKG cotransporters in the BBMV had a lower substrate affinity as compared with those in the BLMV. The transport systems in BLMVs showed a similar Km but different Vmax between the outer cortex (Km: 34 uM, Vmax: 3.3 nmol/mg protein/10s) and outer medulla (Km: 37, Vmax: 1.8). On the other hand, the systems in BBMVs were different in both Km and Vmax between the outer cortex (Km: 194, Vmax: 3.3) and outer medulla (Km: 89, Vmax: 1.7). CONCLUSION: The findings suggest that both axial and apical to basolateral heterogeneity of the Na-alphaKG cotransport system in proximal tubules may be due to a physiological adaptation to efficiently utilize alphaKG in the kidney.

Transport of Tetraethylammonium in Renal Cortical Endosomes of Cadmium-Intoxicated Rats

Effects of cadmium (Cd) intoxication on renal endosomal accumulation of organic cations (OC ) were studied in rats using 14C-tetraethylammnium (TEA) as a substrate. Cd intoxication was induced by s.c. injections of 2 mg Cd/kg/day for 2-3 weeks. Renal cortical endosomes were isolated and the endosomal acidification (acridine orange fluorescence change) and TEA uptake (Millipore filtration technique) were assessed. The TEA uptake was an uphill transport mediated by H /OC antiporter driven by the pH gradient established by H -ATPase. In endosomes of Cd-intoxicated rats, the ATP-dependent TEA uptake was markedly attenuated due to inhibition of endosomal acidification as well as H /TEA antiport. In kinetic analysis of H /TEA antiport, Vmax was reduced and Km was increased in the Cd group. Inhibition of H /TEA antiport was also observed in normal endosomes directly exposed to free Cd (but not Cd-metallothionein complex, CdMt) in vitro. These data suggest that during chronic Cd exposure, free Cd ions liberated by lysosomal degradation of CdMt in proximal tubule cells may impair the endosomal accumulation of OC by directly inhibiting the H /OC antiporter activity and indirectly by reducing the intravesicular acidification, the driving force for H /OC exchange.

Renal Tubular Acidosis in Cadmium-Intoxicated Rats

Effect of cadmium (Cd) intoxication on renal acid-base regulation was studied in adult male Sprague-Dawley rats. Cd intoxication was induced by subcutaneous injections of CdCl2 at a dose of 2 mg Cd/kg/day for 3-4 weeks. In Cd-intoxicated animals, arterial pH, PCO2 and plasma bicarbonate concentration decreased, showing a metabolic acidosis. Urine pH and urinary bicarbonate excretion increased and titratable acid excretion decreased with no change in ammonium excretion. In renal cortical brush-border membrane vesicles derived from Cd-exposed animals, the Na /H antiporter activity was significantly attenuated. These results indicate that chronic exposures to Cd impair the proximal tubular mechanism for H secretion (i.e., Na /H antiport), leading to a metabolic acidosis.

Cadmium inhibition of renal endosomal acidification

Chronic exposure to cadmium (Cd) results in an inhibition of protein endocytosis in the renal proximal tubule, leading to proteinuria. In order to gain insight into the mechanism by which Cd impairs the protein endocytosis, we investigated the effect of Cd on the acidification of renal cortical endocytotic vesicles (endosomes). The endosomal acidification was assessed by measuring the pH gradient-dependent fluorescence change, using acridine orange or FITC-dextran as a probe. In renal endosomes isolated from Cd-intoxicated rats, the Vmax of ATP-driven fluorescence quenching (H -ATPase dependent intravesicular acidification) was significantly attenuated with no substantial changes in the apparent Km, indicating that the capacity of acidification was reduced. When endosomes from normal animals were directly exposed to free Cd in vitro, the Vmax was slightly reduced, whereas the Km was markedly increased, implying that the biochemical property of the H -ATPase was altered by Cd. In endosomes exposed to free Cd in vitro, the rate of dissipation of the transmembrane pH gradient after H -ATPase inhibition appeared to be significantly faster compared to that in normal endosomes, indicating that the H -conductance of the membrane was increased by Cd. These results suggest that in long-term Cd-exposed animals, free Cd ions liberated in the proximal tubular cytoplasm by lysosomal degradation of cadmium-metallothionein complex (CdMT) may impair endosomal acidification 1) by reducing the H -ATPase density in the endosomal membrane, 2) by suppressing the intrinsic H -ATPase activity, and 3) possibly by increasing the membrane conductance to H+ ion. Such effects of Cd could be responsible for the alterations of proximal tubular endocytotic activities, protein reabsorption and various transporter distributions observed in Cd-exposed cells and animals.

Effect of Regular Exercise on Platelet Cytoplasmic Calcium during Treatmill Exercise in Healthy Young Males

BACKGROUND AND OBJECTIVES: Regular aerobic exercise plays a role in the prevention of cardiovascular diseases, but the risk of primary cardiac arrest transiently increases during vigorous exercise, particularly in sedentary men. There has been a controversy regarding the effect of exercise on platelet functional behavior, which is related to the pathogenesis of coronary ischemia. We evaluated the cytoplasmic free calcium concentration of platelets in healthy men after treadmill exercise. MATERIALS AND METHOD: Five physically active (group I: age, 24.2+/-2.3 years) and five sedentary men (group II: age, 22.4+/-1.4 years) were included in this study. Platelet calcium was measured with fluorescent dye, quin2 before and after treadmill exercise. RESULTS: Platelet calcium levels were increased from 86.8+/-11.8 nM to 128.8+/-15.0 nM in group I (p<0.05) and from 102.6+/-14.4 nM to 162.4+/-26.5 nM in group II (p<0.05) immediately after exercise. Thereafter, platelet calcium levels were decreased in group I, but continuously increased in group II. At fifteen minutes after exercise, platelet calcium concentrations were significantly higher than baseline values in group II (205.8+/-53.9 nM vs 102.6+/-14.4 nM: p<0.05), but not in group I (115.2+/-15.7 nM vs 86.8+/-11.8 nM). CONCLUSION: Cytoplasmic free calcium concentration in platelets were continuosly increased after treadmill exercise in sedentary men but not in physically active men.

Effect of Regular Exercise on Platelet Cytoplasmic Calcium during Treatmill Exercise in Healthy Young Males

BACKGROUND AND OBJECTIVES: Regular aerobic exercise plays a role in the prevention of cardiovascular diseases, but the risk of primary cardiac arrest transiently increases during vigorous exercise, particularly in sedentary men. There has been a controversy regarding the effect of exercise on platelet functional behavior, which is related to the pathogenesis of coronary ischemia. We evaluated the cytoplasmic free calcium concentration of platelets in healthy men after treadmill exercise. MATERIALS AND METHOD: Five physically active (group I: age, 24.2+/-2.3 years) and five sedentary men (group II: age, 22.4+/-1.4 years) were included in this study. Platelet calcium was measured with fluorescent dye, quin2 before and after treadmill exercise. RESULTS: Platelet calcium levels were increased from 86.8+/-11.8 nM to 128.8+/-15.0 nM in group I (p<0.05) and from 102.6+/-14.4 nM to 162.4+/-26.5 nM in group II (p<0.05) immediately after exercise. Thereafter, platelet calcium levels were decreased in group I, but continuously increased in group II. At fifteen minutes after exercise, platelet calcium concentrations were significantly higher than baseline values in group II (205.8+/-53.9 nM vs 102.6+/-14.4 nM: p<0.05), but not in group I (115.2+/-15.7 nM vs 86.8+/-11.8 nM). CONCLUSION: Cytoplasmic free calcium concentration in platelets were continuosly increased after treadmill exercise in sedentary men but not in physically active men.

Changes in phosphate transporter activity evaluated by phosphonoformic acid binding in cadmium-exposed renal brush-border membranes

Direct exposure of renal tubular brush-border membranes (BBM) to free cadmium (Cd) causes a reduction in phosphate (Pi) transport capacity. Biochemical mechanism of this reduction was investigated in the present study. Renal proximal tubular brush-border membrane vesicles (BBMV) were isolated from rabbit kidney outer cortex by Mg precipitation method. Vesicles were exposed to 50~200 muM CdCl2 for 30 min, then the phosphate transporter activity was determined. The range of Cd concentration employed in this study was comparable to that of the unbound Cd documented in renal cortical tissues of Cd-exposed animals at the time of onset of renal dysfunction. The rate of sodium-dependent phosphate transport (Na+-Pi cotransport) by BBMV was determined by 32P-labeled inorganic phosphate uptake, and the number of Na+/-Pi cotransporters in the BBM was assessed by Pi-protectable 14C-labeled phosphonoformic acid ((14C)PFA) binding. The exposure of BBMV to Cd decreased the Na+-Pi cotransport activity in proportion to the Cd concentration in the preincubation medium, but it showed no apparent effect on the Pi-protectable PFA binding. These results indicate that an interaction of renal BBM with free Cd induces a reduction in Na+-Pi cotransport activity without altering the carrier density in the membrane. This, in turn, suggest that the suppression of phosphate transport capacity (Vmax) observed in Cd-treated renal BBM is due to a reduction in Na+-Pi translocation by existing carriers, possibly by Cd-induced fall in membrane fluidity.

Increased Activation of Platelet Glycoprotein IIb/IIIa in Hypercholesterolemic Patients

BACKGROUND: Platelet function is directly influenced by lipoproteins, and platelets from hypercholesterolemic patients display increased reactivity which is related to initiation, progression, and development of thromboembolic complications in atherosclerosis. But the exact mechanism of this effect is unclear. METHODS: In this study, total and activated numbers of platelet glycoprotein (Gp) IIb/IIIa were evaluated in twenty patients (7 men; age, 55.4+/-8.7 years) with hypercholesterolemia (plasma total cholesterol level over 240 mg/dL and normal triglyceride level) and twenty one subjects (8 men; 51.1+/-13.7 years) with normal plasma cholesterol and triglyceride levels. Flow cytometry was used to detect the binding of fluorescein isothiocyanate (FITC)-conjugated anti-CD41 or PAC1 to platelet Gp IIb/IIIa in whole blood. When whole blood was incubated with PAC1, platelets were also activated with adenosine diphosphate (ADP) or thrombin. RESULTS: PAC1 was more bound to unstimulated platelets from patients with hypercholesterolemia (p<0.005), and binding of PAC1 correlated significantly with plasma total cholesteol (r=0.48, p=0.002) and LDL-cholesterol (r=0.47, p=0.002) levels. Binding of PAC1 to unstimulated platelets increased as binding of anti-CD41 increased (r=0.40, p=0.01). On multivariate linear regression analysis, plasma total cholesterol level and binding of anti-CD41 were independent variables that determined binding of PAC1. After ADP- or thrombin-stimulation, binding of PAC1 to platelets and percentage of antibody positive cells were also greater in patients with hypercholesterolemia (p<0.05). There was a significant positive correlation between mean platelet volume and binding of anti-CD41 to unstimulated platelets (r=0.46, p<0.0050), but the latter was not different between hypercholesterolemia and control groups. CONCLUSION: Unstimulated platelets from patients with hypercholesterolemia had similar total number of Gp IIb/IIIa to those from control subjects, but had more activated Gp IIb/IIIa. After ADP- or thrombin-stimulation, platelet Gp IIb/IIIa was also more activated under hypercholesterolemia.

Effect of cisplatin on Na+/H+ antiport in the OK renal epithelial cell line

Cis-diamminedichloroplatinum II (cisplatin), an effective antitumor agent, induces acute renal failure by unknown mechanisms. To investigate direct toxic effects of cisplatin in the renal proximal tubular transport system, OK cell line was selected as a cell model and Na+/H+ antiport activity was evaluated during a course of cisplatin treatment. The cells grown to confluence were treated with cisplatin for 1 hour, washed, and incubated for up to 48 hours. At appropriate intervals, cells were examined for Na+/H+ antiport activity by measuring the recovery of intracellular pH (pHi) after acid loading. Cisplatin of less than 50 muM induced no significant changes in cell viability in 24 hours, but it decreased the viability markedly after 48 hours. In cells exposed to 50 muM cisplatin for 24 hours, the Na+-dependent pHi recovery (i.e., Na+/H+ antiport) was drastically inhibited with no changes in the Na+-independent recovery. Kinetic analysis of the Na+-dependent pHi recovery indicated that the Vmax was reduced, but the apparent Km was not altered. The cellular Na+ and K+ contents determined immediately before the transport measurement appeared to be similar in the control and cisplatin group, thus, the driving force for Na+-coupled transport was not different. These results indicate that cisplatin exposure impairs the Na+/H+ antiport capacity in OK cells. It is, therefore, possible that in patients treated with a high dose of cisplatin, proximal tubular mechanism for proton secretion (hence HCO3- reabsorption) could be attenuated, leading to a metabolic acidosis (proximal renal tubular acidosis).

Renal handling of sodium and potassium in cadmium exposed rats

Effects of cadmium exposure on renal Na+ and K+ transports were studied in rats. During the course of cadmium treatment (2 mg Cd/kg/day, s.c. injections for 3 weeks) renal tubular transports of Na+ and K+ were evaluated by lithium clearance technique. During the early phase (first week) of cadmium treatment, urinary Na+ excretion decreased drastically and this was due to an increased Na+ reabsorption both in the proximal and distal nephrons. During the late phase (third week) of cadmium treatment, filtered Na+ load was decreased by reduction in GFR, but the renal Na+ excretion returned to the control level due to impaired Na+ transport in the proximal tubule. Urinary excretion of K+ did not change during the early phase, but it rose markedly during the late phase of cadmium treatment. These results indicate that a light cadmium intoxication induces a Na+ retention, and a heavy intoxication results in a K+ loss. Possible mechanisms for these changes are discussed.

Changes in renal brush-border sodium-dependent transport systems in gentamicin-treated rats

To elucidate the mechanism of gentamicin induced renal dysfunction, renal functions and activities of various proximal tubular transport systems were studied in gentamicin-treated rats (Fisher 344). Gentamicin nephrotoxicity was induced by injecting gentamicin sulfate subcutaneously at a dose of 100 mg/kg cntdot day for 7 days. The gentamicin injection resulted in a marked polyuria, hyposthenuria, proteinuria, glycosuria, aminoaciduria, phosphaturia, natriuresis, and kaliuresis, characteristics of aminoglycoside nephropathy. Such renal functional changes occurred in the face of reduced GFR, thus tubular transport functions appeared to be impaired. The polyuria and hyposthenuria were partly associated with a mild osmotic diuresis, but mostly attributed to a reduction in free water reabsorption. In renal cortical brush-border membrane vesicles isolated from gentamicin-treated rats, the Na+ gradient dependent transport of glucose, alanine, phosphate and succinate was significantly attenuated with no changes in Na+/-independent transport and the membrane permeability to Na+. These results indicate that gentamicin treatment induces a defect in free water reabsorption in the distal nephron and impairs various Na+/-cotransport systems in the proximal tubular brush-border membranes, leading to polyuria, hyposthenuria, and increased urinary excretion of Na+ and other solutes.

Effect of cisplatin on sodium-dependent hexose transport in LLC-PK-1 renal epithelial cells

Cis-dichlorodiammine platinum II (Cisplatin), an effective chemotherapeutic agent, induces acute renal failure by unknown mechanisms. To investigate direct toxic effects of cisplatin on the renal proximal tubular transport system, LLC-PK-1 cell line was selected as a cell model and the sugar transport activity was evaluated during a course of cisplatin treatment. Cells grown to confluence were treated with cisplatin for 60 min, washed, and then incubated for up to 5 days. At appropriate intervals, cells were tested for sugar transport activity using alpha-methyl-D-(14C)glucopyranoside (AMG) as a model substrate. In cells treated with 100 micrometer cisplatin, the AMG uptake was progressively impaired after 3 days. The viability of cells was not substantially changed with cisplatin of less than 100 micrometer, but it decreased markedly with 150 and 200 micrometer. In cisplatin-treated cells, the Na+/-dependent AMG uptake was drastically inhibited with no change in the Na+/-independent uptake. Kinetic analysis indicated that Vmax was suppressed, but Km was not altered. The Na+/-dependent phlorizin binding was also decreased in cisplatin-treated cells. However, the AMG efflux from preloaded cells was not apparently retarded by cisplatin treatment. These data indicate that the cisplatin treatment impairs Na+/-hexose cotransporters in LLC-PK-1 cells and suggest strongly that defects in transporter function at the luminal plasma membrane of the proximal tubular cells constitute an important pathogenic mechanism of cisplatin nephrotoxicity.

pH-Temperature Dependence of Actomyosin Superprecipitation

The effect of temperature on the pH-dependence of actomyosin superprecipitation was studied, using actomyosin extracted from the rabbit and frog skeletal muscle tissues. The pH optima of superprecipitation was rather broad in both the rabbit and frog actomyosin. In the frog, superprecipitation measured at 16-42 degrees C was relatively independent of pH variations between 6.7 to 8.5, but it was significantly inhibited at pHs outside of this range, showing a sharp inflection of the curve. The pH at the inflection point was inversely proportional to the incubation temperature, but the (OH-)/(H+) ratio at the inflection point was not changed with temperature. The log (OH-)/(H+) was approximately -0.6 on the acidic side and 3.16 on the alkaline side. Similarly, superprecipitation of the frog actomyosin was virtually independent of the medium pH of the intermediate range (approximately 6.0-8.5); but it was drastically inhibited at pHs below or above this range, thus revealing a sharp inflection of the curve. Again, the pH at the inflection point changed inversely with temperature, preserving a constant (OH-)/(H+) ratio. The log (OH-)/(H+) ratio at the inflection point was approximately -2 on the acidic side and 3.5 on the alkaline side. The above pH effects were not associated with irreversible protein damage or with the changes in buffer species. These results strongly suggest that suppression of the superprecipitation of rabbit and frog actomyosin gels, at a low and high pH, be due to alterations in the fractional dissociation of histidine-imidazole and cysteine-SH groups, respectively.

Role of Sodium Ion in Renal Transport of p-Aminohippurate in vitro

The effect of sodium on p-aminohippurate (PAH) transport kinetics was studied in isolated rat kidney slices in an attempt to define the role of sodium ion in renal organic acid transport. 1. In normally metabolizing renal slices, Na+ increased the Vmax of PAH influx without changing the Michaelis constant (Km). On the other hand, the effIux of preaccumulated PAH was reduced as the Na+ concentration increased. 2. In metabolically impaired renal slices, Na+ had no apparent effect on the influx and efflux of PAH. These results may indicate that Na+ is important for the energy transducing reaction in the PAH transport process.

Biochemical and Physiological Characteristics of Ca-ATPase System of Rat Liver Mitochondria with Special Attention to the Effects of pH and Temperature

The activity of Mg++-dependent, Ca++-activated adenosine triphosphatase (Ca-ATPase) of rat liver mitochondria was studied at varying medium compositions, pH and temperatures. The enzyme system was characteristically sensitive to Ca++ concentration with a KmCa of approximately 0.06 mM. The optimal concentration of Mg was about l mM, above which the enzyme activity was progressively inhibited. The inhibitory effect of high Mg++ concentrations appeared to be due to the alteration of the Mg++/ATP ratio. Variations in the Mg++/ATP ratio affected Vmax but not the KmATP. The pH optimum for enzyme activity increased as the incubation temperature decreased, but the optimal OH-/H+ ratio of the medium was constant at around 0.1, regardless of temperature. The activity of the enzyme was not affected by La# (0.01-1 mM) and Ruthenium red (2.5-10.0 microM). These results indicate that 1) the enzymatic characteristics of the Ca-ATPase system in the rat liver mitochondria is typical of those from other tissue preparations, 2) the enzyme system maintains the most effective catalytic conformation at a fixed level of OH-/H+ ratio of 0.1 when the temperature changes, and 3) the enzyme system may not play a role in the physiological transport of Ca++ in mitochondria.

Changes in Rena1 Na-K-ATPase Activity and PAH Transport Kinetics in Uninephrectomized Rats and Cold Exposed Hamsters

Renal Na+, K+-activated adenosinetriphosphatase (Na-K-ATPase) activity and the p-aminohippurate (PAH) transport kinetics were studied in uninephrectomized rats and cold exposed hamsters. In rats, the specific activity of renal Na-K-ATPase increased by approximately 50% in one week after uninephrectomy and remained more or less constant during the next three weeks. The capacity (Jmax) of PAH influx into the renal cortical slice was sharply increased in one week after nephrectomy, but after which it returned to the control level. In cold exposed hamsters, the specific activity of renal Na-K-ATPase did not increase until 48 days of cold exposure at which time it reached approximately 50% above the control level. On the other hand, the Jmax of PAH influx increased by about 80% in 10 days of co1d exposure and somewhat declined thereafter. These results suggest that PAH active transport in the renal slice is not ratelimited by the activity of Na-K-ATPase under physiological conditions.

The Effect of Halothane-induced Hypotention on the Kidney / 대한마취과학회지

Hypotensive anesthesia is widely used in an operation since 1) it minimizes bleeding and provides a good operation field, 2) it prevents massive hemorrhage in an operation which otherwise involves a large amount of b1ood loss and 3) it is useful in an operation for hypertensive patients. This procedure also involves a number of risks such as delayed awakening, reactionary bleeding, decrease in urine output and tissue hypoxia. The most dangerous complication can be hypoxia especially in the vital organs. 1t is therefore necessary to treat dehydration or blood loss before hypotensive anesthesia is induced. Since hypotensive anesthesia was introduced by Gardner (1947) for an operation of olfactory groove meningioma, various methods of deliberate hypotension have been developed. The most common method of hypotensive anesthesia in the present day is to use drugs, such as trmetaphan, nitroprusside and halothane. The effect of hypotensive anesthesia an various physiological functions of animals have been investigated is the past. Giffiths and Gillies (1948) reported that, in the hypotension induced by sympathectomy, an arterial systolic pressure of 32 mmHg is the minimum to overcome peripheral resistance. Chung (19743 observed in the halothane-induced hypotensive dogs that a systolic pressure of 30mmHg was required to assure adequate cerebral oxygenation. In the present study, we have investigated the effect of halothanie-induced hypotension on the renal function of dogs. The arterial systolic pressure was successively reduced to 60 and 30mmHg for 30min. each, and changes is various renal functions were studied during 100min. of the recovery period. The results are summarized as follows: 1. The systolic blood pressure was completely reeovered 80min. after the cessation of halothane inhalation. 2. Average renal blood flow Cestimated by Cppe) and glomerular filtration rate (estimated by Cg) during the first 20min. of the recovery phase were 26% ind 45% of the control level. However after 100min. of thy recovery period, repal blood flow was recovered to 63% and glomerular filtration rate to 74%of the control leveL 3. Average urine flow during the first 20min. of that recovery phase was approximately 40% of the control. 4. U/P osm. ratio was reduced to 90% the control level during the first 20min. of recovery, but it exceeded the control value after 20min. of recovery. 5. Thero was only 24% of the-tml value in the first 20min. of receavery phese, but there- after it gradually returned to the control level. 6. FEH2O (fractional excretion of N2) was and significantly changed- by halothane inhalation although there was a tendency to slight reduction at the beginning of the recovery phase. 7. FEK was 47% of the control value dqring ghe initial phase of recovery, but it returned to the control level after 40min. of the recovery period. 8. FE, and FEH2O were reduced to 56% and 50% of the control level after the hypotensive period, but returned to 70% and 82% of the control level after 40min. of the recovery period. These results indicate that although the systemic blood pressure completely recovered after halothane-induced hypotension, renal hemodynamics are not completely recovered with 100min. of the recovery period. However renal functions are mostly reversible, suggesting that halathane -induced hypotension did not induce irreversible damage of renal tissue.

The Effect of Amphotericin B on Erythrocyte Volume and Cation Content

The effects of amphotericin B, an antifungal antibiotic, on erythrocyte volume and cation permeability were investigated by measuring the hematocrit, cell volume, cation content, fragility and osmotic behavior in rat erythrocytes, in vitro. 1. When erythrocytes were incubated in a Ringer solution containing amphotericin B (5-25 microgram/ml) the hematocrit and the cell volume increased, the effect being proportional to the concentration of the drug and the incubation time period. 2. Amphotericin B increased the Na content and decreased the K content of the erythrocyte. In normal Ringer solution (NaCl-Ringer)containing amphotericin B the magnitude of cellular Na gain was greater than that of K loss. Therefore, the total cellular cation content increased. On the other hand, when cells were incubated in the amphotericin B containing Ringer solution in which NaCl was replaced by Na2SO4 (Na2SO4-Ringer) the magnitude of cellular K loss exceeded that of cellular Na gain. Consequently, the total cellular cation content was reduced. 3. Amphotericin B increased cell volume (hematocrit) when erythrocytes were incubated in the Na2SO4-Ringer solution. 4. The fragility of erythrocytes increased when cells were preincubated in the amphotericin B containing normal Ringer solution, whereas it decreased in tile cells preincubated in the amphotericin B containing Na2SO4-Ringer solution. 5. The cell volume was linearly related to the reciprocal of medium osmolality(200 to 900 mOsm/kg H2O) in both NaCl-and Na2SO4-Ringer solutions, and the linearity was not altered by amphotericin B. The antibiotic did not change the slope of the correlation line (V vs. 1/OSM). It, however, increased the intercept of the line with the ordinate in normal Ringer solution and decreased that in the Na2SO4-Ringer solution. These results indicate that amphctericin B alters the cell volume by changing the permeability of Na and K across the membrane.