Delayed haemolytic action of palytoxin. General characteristics

1. Palytoxin is a haemolysin. The erythrocytes from various species can be classified into a sensitive and a hardly sensitive group. The former contain potassium as their main inside cation and are arranged according to their sensitivity as hog greater than or equal to rat, mouse greater than rabbit greater than guinea-pig greater than man. The latter, comprising those from sheep and cattle, have sodium as their main inside cation. In addition, chicken erythrocytes are relatively insensitive. 2. Haemolysis of rat erythrocytes is preceded by a lag period of 1--2 h. With increasing temperature the haemolysis proceeds more quickly but reaches the same final range between 25 and 42 degrees C. The pH optimum in Britton-Robinson buffer supplemented with saline is between 7 and 8. Washing off palytoxin during the prelytic period reduces the haemolytic power. 3. The sensitivity of rat erythrocytes decreases with increase of osmolarity between 235 and 415 mosM. Accordingly, their osmotic resistance is lowered by palytoxin in a concentration-dependent manner. 4. With both rat and sheep erythrocytes, potassium loss by far precedes the haemolysis due to palytoxin. Potassium loss is measurable already after 1 min and increases with time. After 2 hours the quotient between the ED50 of haemolysis and that of potassium loss is around 200. Thus palytoxin is an unusually strong but slow haemolysin of the osmotic type. The extreme prelytic potassium loss and the correlation between susceptibility and potassium content of erythrocytes points towards the relevance of ionic fluxes.     

Changes in erythrocyte permeability due to palytoxin as compared to amphotericin B

Palytoxin causes within minutes a temperature-dependent K+ loss from human and rat erythrocytes which is followed within hours by haemolysis. It decreases the osmotic resistance in a concentration-dependent manner, so that osmotic influences are negligible for K+ release but considerable in haemolysis. External K+ inhibits the haemoglobin release and Rb+ inhibits the release of K+ and haemoglobin. Ca2+ (over 20 microM) and borate (over 5 microM) enhance the loss of K+ and haemoglobin. With both Ca2+ and borate present, the efficacy of palytoxin is raised about 10 000-fold. Under these conditions, about 15 palytoxin molecules per human cell trigger a 50% K+ loss over a wide range of cell concentrations. The palytoxin effect is reversible. After depletion from K+ by low concentrations of palytoxin, human cells can be refilled with K+ and resealed. The pores formed by palytoxin are small. They allow the entrance of Na+ and choline, whereas inositol is largely excluded and Ca2+, as well as sucrose and inulin, are completely excluded. Amphotericin B resembles palytoxin in its ability to cause a considerable prelytic K+ loss and to form small pores. However, it is about 1000-times weaker than palytoxin, is not inhibited by K+ or Rb+, is not activated by Ca2+ or borate, and has a negative temperature dependence. Thus palytoxin represents a novel type of cytolysin.     

The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine

(1) Using the phosphatidylcholine specific transfer protein from bovine liver, native phosphatidylcholine from intact human erythrocytes was replaced by a variety of different phosphatidylcholine species without altering the original phospholipid and cholesterol content. (2) The replacement of native phosphatidylcholine by the disaturated species, 1,2-dipalmitoyl- and 1,2-distearoylphosphatidylcholine, proceeded at a low rate and extensive replacement could only be achieved by repeatedly adding fresh donor vesicles. The replacement by disaturated molecules was accompanied by a gradual increase in osmotic fragility of the cells, finally resulting in hemolysis when 40% of the native PC had been replaced. Up to this lytic concentration, the replacement did not affect the permeability of the membrane for potassium ions. (3) Essentially, all of the PC in the outer monolayer of the membrane could be replaced by 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine. These replacements did not alter the osmotic fragility of the cells, nor the K⁺ permeability of the membrane. (4) Increasing the total degree of unsaturation of the phosphatidylcholine species modified the properties of the membrane considerably. Replacement by 1,2-dilinoleoylphosphatidylcholine resulted in a progressive increase in osmotic fragility and hemolysis started to occur after 30% of the native PC had been replaced by this species. K⁺ permeability was found to be slightly increased in this case. Cells became leaky for K⁺ upon the introduction of 1-palmitoyl-2-arachidonoylphosphatidylcholine in the membrane. The increased permeability was also reflected by an apparent increase in the resistance of the cells against osmotic shock. (5) The conclusions to be drawn are that (i) 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine are species which fit most optimally into the erythrocyte membrane; (ii) loss of membrane stability results from an increase in the degree of saturation of phosphatidylcholine ($\text{unsaturation index}\text{> 0.5}$) and (iii) the permeability is enhanced by increasing the content of highly unsaturated species ($\text{unsaturation index}\text{> 1.0}$).     

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

Permeability changes of erythrocytes and liposomes by 5-(n-alk(en)yl) resorcinols from rye

$\text{5-(n-}\text{Alk(en)yl}\text{)}$ resorcinols can induce potassium release from liposomes and erythrocytes. The results suggest that $\text{5-(n-}\text{pentyl)resorcinol}$ can induce a specific permeability to protons as well as to potassium and other small molecules. The highest permeability changes were found in the presence of $\text{5-(n-}\text{pentadecyl)resorcinol}$ and alkenyl resorcinols. Orcin and resorcin were without effect. The size of permeant as investigated by turbidity measurements indicated that Ca²⁺ and Mg²⁺ cannot pass through the alkyl resorcinol-modified membrane but can pass through the alkenyl resorcinol-modified membrane. It was observed that alkenyl resorcinol at a concentration of 15 μM induced not only potassium release but also lysis of erythrocytes.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Rapid loss of δ-aminolevulinic acid dehydratase activity in primary cultures of adult rat hepatocytes: A new model of zinc deficiency

Nonproliferating cultures of adult rat hepatocytes were found to lose 60–70% of cell-associated zinc during their first 24 h of incubation in standard, serum-free medium. The loss of zinc was accompanied by a profound loss (95%) in the activity of the zinc metalloenzyme, δ-aminolevulinic acid dehydratase, as well as a loss (>85%) in the cellular content of immunoreactive δ-aminolevulinic acid dehydratase protein. Restoration of cellular zinc content by the addition of zinc to the culture medium partially prevented the losses of both δ-aminolevulinic acid dehydratase activity and immunoreactive protein. Since the spontaneous, selective loss of cellular zinc appears to have specific effects on a relevant hepatic function, this culture system constitutes a novel $\text{in}$$\text{vitro}$ model of zinc deficiency in mature liver.     

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes

Ouabain-sensitive Na⁺ and K⁺ fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl₂ and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. p] It was found that an increased cellular pH reduced the rates of active transport of Na⁺ and K⁺ without significantly altering the ratio of pumped $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$. This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between ?10 and +60 mV at constant internal pH did not affect the rates of active transport of Na⁺ or K⁺.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.     

A new type of enzyme electrode: The ascorbic acid eliminator electrode

A new type of enzyme electrode has been developed for $\text{in}$$\text{vivo}$ electrochemical measurements which allows discrimination between ascorbic acid and catecholamines and their metabolites. The electrode employs the enzyme ascorbic acid oxidase held between the voltammetric electrode and solution by a dialysis membrane or immobilized on the outer surface of serous membrane from rat small intestine. The electrode gives linear calibration curves for all catecholamines and metabolites independent of any ascorbic acid concentrations significant in physiological measurements. The electrode has been tested in brain slice measurements and shown to respond to releases of catecholamines initiated by potassium ion stimulation.     

Interaction of dichlone with human erythrocytes. I. Changes in cell permeability

The uptake of the fungicide dichlone (2,3-dichloro-1,4-naphthoquinone) by human erythrocytes was extremely rapid, reaching a maximum within 5 min of treatment. Most of the dichlone taken up was present in the interior of the cell; only a small fraction of the pesticide (less than 5%) was bound to the cell membrane. Dichlone (3 · 10^?5M-10^?4M) induced a rapid loss of intracellular potassium from the erythrocytes; the leakage of K⁺ varied with the fungicide concentration as well as with cell concentration. Pretreatment of the cells with glutathione was able to reduce potassium loss. Cells exposed to dichlone showed increased osmotic fragility. Dichlone also inhibited Na⁺-K⁺ ATPase, which is associated with active ion transport. However, the leakage of potassium in dichlone-treated cells does not appear to be related to the interference with active ion transport. An extensive loss of potassium within a relatively short time after treatment suggests that dichlone produces its effect by increasing passive cation permeability, probably as a result of direct action on the membrane structure. Dichlone was able to induce hemolysis, but only at concentrations higher than those which resulted in K⁺ loss. The loss of hemoglobin appeared to be mainly due to osmotic swelling of the treated cells. Exposure of red cells to dichlone also resulted in a rapid and extensive formation of methemoglobin as well as a denaturation of hemoglobin. Thus, dichlone not only may be capable of lowering the capacity of erythrocytes to transport oxygen but also alters their permeability.     

Effect of lysophosphatidylcholine on salt permeability through the erythrocyte membrane under haemolytic conditions

Human erythrocytes were incubated in haemolytic salt or sucrose media and the amount of potassium and haemoglobin released were monitored. In hypotonic NaCl and KCl solutions potassium release and haemolysis increased with time showing that the cell membrane had been injured and became permeable to intra- and extracellular cations which, due to intracellular haemoglobin, causes water influx and continuous haemolysis. Both potassium release and haemolysis remained, however, at their 2-minute level in the presence of LPC. Thus, LPC could reseal the membrane and prevent continuous salt fluxes. It protected erythrocytes from hypotonic haemolysis and the protection was more efficient in NaCl than in sucrose media. This suggests that the increase in the critical volume of erythrocytes caused by LPC occurs both in electrolyte and sucrose media, and the additional protection observed in electrolyte media is due to the resealing of the injured cell membrane by LPC. The repairing mechanism was mediated via the membrane lipids or integral proteins, since the time-course of haemolysis of erythrocytes swollen in NaCl media at the spectrin-denaturing temperature of 49.5 degrees C was similar to that at room temperature with and without LPC. LPC did not protect erythrocytes from colloid osmotic haemolysis caused by ammonia influx in an isotonic NH4Cl medium, but protected the cells from colloid osmotic haemolysis caused by sodium influx through nystatin-channels in NaCl media without any area or volume increase. Hence, LPC could not prevent ammonia influx through the lipid bilayer, but suppressed sodium influx through nystatin-channels presumably via LPC interference with cholesterol.     

The permeability of the lysosomal membrane to small ions

The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K⁺ in the presence of valinomycin, and cation influx was coupled to an efflux of H⁺ using the protonophore $\text{3-tert-}\text{butyl-5,2′-dichloro-4′-nitrosalicilylanilide}$. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be $\text{SCN}{}^{\mathrm{?}}\text{> I}{}^{\mathrm{?}}\text{> CH}{}_3\text{COO}{}^{\mathrm{?}}\text{> Cl}{}^{\mathrm{?}}\text{≈ HCO}{}^{\mathrm{?}}{}_3\text{≈ P}{}_{\mathrm{i}}\text{> SO}{}_4{}^{\mathrm{2?}}$ and that to cations $\text{Cs}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> H}{}^{\mathrm{+}}$. These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.     

The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes

Summary The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca²⁺ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca²⁺ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca²⁺ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca²⁺. These data indicate that the repeat domain of haemolysin is responsible for Ca²⁺-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca²⁺ in haemolysis is presented.     

On the mechanism of energy-dependent contraction of swollen mitochondria.

Electrophoretic cation permeability, as estimated by rates of passive swelling of mitochondria suspended in Na+ and K+ nitrate, increases with increasing temperature and elevated pH and is inhibited by Mg+². Mitochondria swollen in Na+ nitrate at 37° and pH 8.2 contract in an energy-dependent reaction. The efficiency of the contraction (absorbance change per O₂ or ATP consumed) decreases with increased electrophoretic cation permeability as established by either elevated pH or addition of gramicidin. Efficiency is increased by Mg+². This inverse relationship between electrophoretic cation permeability and efficiency of contraction is compatible with an osmotic contractile mechanism which depends on the $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger present in the mitochondrial membrane.     

The accumulation ratio of K+, Na+, Ca2+ and tetrapropylammonium in steady-state Mitochondria.

The accumulation ratio of a permeant cation under steady-state conditions after active uptake, is defined as: ($\text{{cat}{}_{\mathrm{i}}\text{}}\text{{cat}{}_0\text{}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>z</mtext>}}$, where Z is the valence of the cation, and {cat_i} and {cat₀} are the internal and external cation activities, respectively. The electrogenic proton pump predicts that the accumulation ratio should be independent of (i) the chemical structure of the cation and (ii) the degree of permeability of the membrane to cations. Furthermore the accumulation ratio should be the same for all permeant cation species simultaneously present. In the present study it is found that under steady-state conditions: (i) the accumulation ratio is not the same for potassium in the presence of valinomycin, for tetrapropylammonium in the presence of tetraphenylboron, and for calcium in the presence of acetate; (ii) the accumulation ratio is not identical for two cations such as potassium and sodium present simultaneously in the presence of gramicidin; (iii) the accumulation ratio is dependent on the external carrier concentration, such as valinomycin or tetraphenylboron. It is concluded that the cation distribution ratios under steady-state conditions are not compatible with the predictions of the electrogenic proton pump model.     

Alanine transport by the isolated perfused rat kidney

L-alanine transport kinetics were examined in the isolated perfused rat kidney (1) using different perfusate concentrations of alanine (PAla) to obtain different filtered loads and (2) under conditions of osmotic diuresis. The transport maximum for alanine (TmAla) was found to be very high relative to $\text{in}$$\text{vivo}$ filtered loads of alanine. The apparent TmAla was dependent on glomerular filtration rate (GFR) and it could be modified by osmotic diuresis. It is suggested that the variation of TmAla with changes in GFR may be the consequence of variations in fractional volume flow through the proximal tubule.