Inverse dependence of binding constants upon albumin concentration. Results for L-tryptophan and three anionic dyes.

The interactions of methyl orange, bromocresol green, 2-(4'-hydroxybenzeneneazo)benzoic acid (HABA) and L-tryptophan with human albumin at pH 7.4 were investigated by equilibrium dialysis at 37 degrees C. Binding characteristics of each of the three dyes were studied by two approaches: (1) variation of total ligand concentration with a single albumin concentration and (2) variation of albumin concentration with a single total ligand concentration. Both approaches gave typical Scatchard plots with negative slope for methyl orange and bromocresol green, with good agreement between the two sets of data for each dye. In contrast, approach (2) gave Scatchard plots with a positive slope for HABA and L-tryptophan, indicating a decrease in the number of binding sites (n) and/or association constant (k) as the albumin concentration increased. This inverse dependence of nk upon albumin concentration for 2-(4'-hydroxybenzeneazo)benzoic acid was mainly due to changes in n which were still observed in the presence of inhibitory chloride ions at pH 5.75. Reasons for this type of binding behaviour are discussed together with general implications for binding studies. The results show 2-(4'-hydroxybenzeneazo)benzoic acid to be a useful ligand for investigation of this problem.

Decreased binding of drugs and dyes to plasma proteins from rats with acute renal failure: effects of ureter ligation and intramuscular injection of glycerol.

1 The decreased binding of drugs and dyes to plasma proteins from male and female rats with acute renal failure has been investigated using equilibrium dialysis at 37 degrees C. 2 Acute renal failure induced by bilateral ligation of the ureters produced a greater than threefold increase in the unbound fraction of o-methyl red relative to normal rat plasma. Unbound dye concentration in plasma from sham-operated control rats was also significantly increased but to a lesser extent. 3 Glycerol-induced acute renal failure produced a significant increase in the unbound fractions of o-methyl red, methyl orange, bromocresol green (BCG), 2-(4'-hydroxybenzeneazo) benzoic acid (HABA), phenytoin and salicylic acid. A marginally significant increase in unbound warfarin concentration was observed. 4 Glycerol-induced renal failure had no effect on total plasma protein concentration and experiments with o-methyl red and salicylic acid indicated that a direct effect of glycerol was not responsible for the diminution of binding. 5 Glycerol-induced acute renal failure, which produced decreases in drug and dye binding similar to those reported for human uraemic plasma, provides a convenient non-surgical animal model for the investigation of this phenomenon.

Pharmacokinetics and biliary excretion of bromosulphophthalein, [3H]-ouabain and [3H]-taurocholic acid in rats with glycerol-induced acute renal failure.

The pharmacokinetics and biliary excretion of bromosulphophthalein (BSP), ouabain and taurocholic acid (TChA) have been studied in rats with glycerol-induced acute renal failure (ARF). In rats with ARF, the hepatic uptake and initial biliary excretion of BSP were decreased. In addition, the rate of BSP conjugation with glutathione by rat liver homogenates was also decreased. This latter change may contribute to the initial decrease in the biliary excretion of BSP. No change was found in the hepatic uptake and biliary excretion of ouabain, but the area under the concentration-time curve was increased and the plasma clearance (Clp) decreased in rats with ARF. This decrease in Clp was not due to reduced renal excretion. The decreased Clp of ouabain in rats with ARF may come from reduced tissue binding and a concomitant decrease in its volume of distribution (Vd). The hepatic handling of TChA appeared unaltered in ARF, but the rate constant for the terminal part of the concentration-time curve (beta) was decreased. This change probably resulted from a large increase in Vd in rats with ARF. It is concluded that the decreased uptake of BSP was not due to a non-specific disturbance of hepatocyte function in ARF because the hepatic handling of ouabain and TChA were unaltered.

Dibromosulphophthalein: its pharmacokinetics and binding to hepatic cytosol proteins in rats with acute renal failure.

1. The pharmacokinetics, biliary excretion and binding of dibromosulphophthalein (DBSP) to plasma proteins and hepatic cytosol proteins have been studied in male rats with glycerol-induced acute renal failure (ARF). 2. The rate constants for hepatic uptake, efflux from liver to plasma and excretion into bile were all significantly decreased in rats with ARF. Furthermore, the plasma clearance of DBSP was also reduced. 3. The initial (0-10 min) and maximum biliary excretion rates of DBSP were both diminished in animals with ARF. The maximum excretion rate occurred between 5-10 min in control rats and 10-15 min in rats with ARF. However, there was no statistically significant change in the percentage dose recovered from bile after 30 min. 4. The plasma-protein binding of DBSP was decreased in rats with ARF and this change was due to a significant reduction in the association constant for the primary binding sites. 5. The binding of DBSP to ligandin (Y protein) was reduced by about 38% in rats with ARF but no change was noted in binding to Z protein. Reduced binding to ligandin was accompanied by decreased total liver glutathione S-transferase (GST) activity and a 36% reduction in the GST activity of ligandin. 6. The results support the contention that altered hepatic handling of cholephilic dyes in rats with ARF may be due to reduced binding to ligandin.

Effect of the adenosine antagonist 8-phenyltheophylline on glycerol-induced acute renal failure in the rat.

8-Phenyltheophylline (8-PT)(10 mg kg-1) or its vehicle(1 ml kg-1) were administered intravenously or intraperitoneally twice daily over 48 h to rats with acute renal failure (ARF) induced by intramuscular (i.m.) injection of glycerol. Rats treated with 8-PT i.v. had significantly lower plasma urea and creatinine levels at 24 and 48 h compared to untreated animals. The vehicle also reduced plasma urea and creatinine when compared to untreated controls. However, plasma urea levels in 8-PT-treated rats were significantly lower than in vehicle-treated animals at 24 and 48 h after both i.v. and i.p. administration. Plasma creatinine concentrations also tended to be lower in the 8-PT-treated group. [3H]-inulin clearance at 48 h after i.m. glycerol was significantly greater in rats dosed i.p. with 8-PT compared to either untreated or vehicle treated rats. Examination of kidneys taken from rats 48 h after i.m. glycerol showed that 8-PT treatment significantly reduced renal damage and kidney weight compared to the untreated or vehicle-treated groups. In a 7 day study all the rats which received 8-PT i.p. survived whilst in the vehicle and untreated groups the mortality rates were 12 and 21% respectively. In a separate series of experiments 8-PT (10 mg kg-1, i.v. or i.p.) was found to antagonize adenosine-induced bradycardia in conscious rats for up to 5 h. There is no clear explanation for the partial protection afforded by the vehicle but it may be related to either its alkalinity or an osmotic effect produced by the polyethylene glycol component. 9 The protective effect of 8-PT in rats with ARF was probably the result of adenosine antagonism.

Amelioration of glycerol-induced acute renal failure in the rat with 8-cyclopentyl-1,3-dipropylxanthine.

1. Previous studies have shown that 8-phenyltheophylline (8-PT), a non-selective antagonist at adenosine A1- and A2-receptors, can ameliorate the severity of glycerol-induced acute renal failure (ARF) in the rat. In the present study we have examined the effects of an antagonist with selectivity for adenosine A1-receptors (8-cyclopentyl-1,3-dipropylxanthine, CPX) on the development of ARF. 2. In the anaesthetised rat 8-PT (4 mg kg-1, i.v.) and CPX (0.1 mg kg-1, i.v.) antagonised adenosine-evoked responses which are thought to be mediated via A1-receptors (bradycardia and decrease in renal blood flow). The agonist dose-ratio produced by CPX was equal to or greater than that found with 8-PT (heart rate and renal blood flow respectively). The hypotensive response to adenosine which is predominantly due to A2-receptor activation was also antagonised by 8-PT, whereas CPX was a much less effective antagonist of this response. 3. Administration of CPX (0.1 mg kg-1, i.v.; twice daily for two days) significantly attenuated the increase in plasma levels of urea and creatinine, the increased kidney weight and the renal tubule damage observed in rats 2 days following induction of ARF with intramuscular glycerol injection. In addition treatment with CPX significantly enhanced the clearances of inulin and p-aminohippurate. 4. After glycerol injection, the mortality rate over 7 days in untreated and vehicle-treated rats was 43% and 21% respectively. In contrast, all animals treated with CPX survived over the 7 day observation period. 5. These results support the suggestion that adenosine is an important factor in the development of ARF and indicate that this effect of the purine is likely to be mediated via an adenosine A1-receptor.

Cardiovascular responses in rats with glycerol-induced acute renal failure.

Autonomic and cardiovascular function were assessed in rats with glycerol-induced acute renal failure (ARF). Rats with ARF had significantly lower mean arterial blood pressure and heart rates and significantly elevated plasma noradrenaline concentrations. The chronotropic responses to right cervical sympathetic and vagal stimulation were diminished in rats with ARF. The pressor and depressor responses to noradrenaline and nitroprusside respectively when expressed as a change in mmHg pressure were significantly reduced in rats with ARF when compared to controls. However, when the depressor responses to nitroprusside were expressed as a percentage fall in basal mean arterial pressure, with the exception of the response to a dose of 10 micrograms kg-1, there were no significant differences between control and uraemic rats. The present findings show that in the rat, changes in cardiovascular responsiveness occur after a brief period of uraemia which are similar to those observed in patients and rats with chronic renal failure.

The diuretic action of 8-cyclopentyl-1,3-dipropylxanthine, a selective A1 adenosine receptor antagonist.

1. The diuretic effect of the selective A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), was investigated in anaesthetized rats. 2. CPX (0.1 mg kg-1, i.v.) produced significant increases in urine flow, and the excretion rate and fractional excretion of both sodium and chloride. By contrast, CPX administration did not result in any significant change in the excretion of potassium. 3. The diuretic effect of CPX was accompanied by a transient increase in inulin clearance although p-amino-hippurate clearance was unaffected, indicating the CPX induced a temporary elevation of glomerular filtration rate but no change in renal blood flow. 4. The fractional excretion of lithium (a marker of delivery of fluid out of the proximal tubule) was also significantly increased by CPX. However, other measures of tubular function derived from lithium clearance indicated that there were no changes in the handling of sodium or water in the distal regions of the nephron. 5. CPX did not significantly alter the relationship between either free water reabsorption or free water clearance and the distal delivery of sodium, which suggests that CPX does not affect the renal concentration/dilution mechanism. 6. The results of this study show that the diuresis and increased excretion of sodium and chloride induced by CPX (0.1 mg kg-1) in the rat, occurs with only transient elevation in glomerular filtration rate and no change in renal blood flow. The primary reason for the diuresis appears to be inhibition of sodium reabsorption in the proximal tubule. Furthermore, the results provide evidence that production and release of endogenous adenosine modifies renal excretory function via stimulation of the A1 receptor subtype.

Amelioration of cisplatin-induced acute renal failure with 8-cyclopentyl-1,3-dipropylxanthine.

1. The effect of the selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), on the development of cisplatin-induced acute renal failure was investigated in the rat. 2. CPX at doses of 0.03, 0.1 and 0.3 mg kg-1, i.v. caused increasing degrees of antagonism of adenosine-induced bradycardia in anaesthetized rats. The magnitude of antagonism was not directly proportional to the increment in dose, but for each dose, it was similar in rats injected with either saline or cisplatin. CPX at a dose of 0.03 mg kg-1 significantly antagonized adenosine-induced bradycardia for up to 2.5 h, while doses of 0.1 and 0.3 mg kg-1 produced significant blockade for periods longer than 5 h. 3. Administration of cisplatin (6 mg kg-1, i.v.) caused acute renal failure characterized by decreased inulin and p-aminohippurate clearances, increased urine volume but decreased excretion of Na+, K+ and Cl- ions and by increased plasma levels of urea and creatinine. Kidney weight was increased in cisplatin-treated rats and renal tubule necrosis occurred. 4. Administration of CPX (0.03 mg kg-1, i.v.; twice daily for two days) to rats given cisplatin did not reduce the severity of the resultant renal failure. However, treatment with 0.1 mg kg-1 CPX attenuated the increases in plasma creatinine/urea levels observed in rats on days 3 and 7 after induction of renal failure. In addition, this dose significantly reduced renal tubule damage and increased inulin and p-aminohippurate clearances. A similar pattern of protection was noted with CPX at a dose of 0.3 mg kg-1 although the increase in inulin clearance was not statistically significant. However, this higher dose of CPX significantly increased Na+ and K+ excretion compared to vehicle-treated rats. 5 CPX at doses of 0.03, 0.1 and 0.3 mgkg- produced blockade of an A1-receptor mediated response i.e. adenosine-induced bradycardia, but only treatment with the higher doses of CPX (0.1 and 0.3mgkg-1) ameliorated nephrotoxicity produced by cisplatin. The lack of any protective effect afforded by the lowest dose of CPX could be a result of its shorter duration of action.6. This study indicates that adenosine plays a significant role in the pathophysiology of cisplatin-induced acute renal failure.

Vascular reactivity in rats with glycerol-induced acute renal failure.

The vascular reactivity of rats with glycerol-induced acute renal failure was assessed in vitro and in vivo. The contractile responses to noradrenaline, angiotensin and potassium chloride of aortic strips and portal vein segments from uraemic rats were significantly smaller than the responses obtained in vessels from control animals. The pressor responses to angiotensin were reduced significantly in rats with acute renal failure when compared to those in control rats. The reduced vascular responses to a range of spasmogens suggests that in this model of renal failure there is a defect in some mechanism of vascular contraction common to all three constrictor agents.

Renal adenosine A1 receptor binding characteristics and mRNA levels during the development of acute renal failure in the rat.

1. The binding characteristics and mRNA levels for renal adenosine A1 receptors were investigated in normal rats and rats with acute renal failure (ARF) induced by either glycerol or HgCl2. 2. Saturation isotherms determined from the binding of [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), a selective adenosine A1 antagonist, to renal membranes of untreated rats gave values of 0.62 nM for the equilibrium dissociation constant (Kd) and 19.9 fmol mg-1 protein for the density of binding sites (Bmax). No saturable binding was observed with [3H]-2-(p-(carboxylethyl)-phenylethylamino)-5'-N-ethylcar box amido adenosine ([3H]-CGS 21680), a selective adenosine A2a agonist. 3. By contrast to time-matched controls, renal membranes obtained from rats 16 and 48 h following the induction of ARF with glycerol, showed statistically significant increases (2-4 fold) in both Bmax and Kd for the binding of [3H]-DPCPX. No significant changes in the binding characteristics of [3H]-DPCPX were noted with membranes from rats 48 h following the production of ARF with HgCl2. 4. Adenosine A1 receptor mRNA levels were significantly elevated 0.5, 16 and 48 h following induction of ARF with glycerol, whilst no change was noted in mRNA levels for beta-actin at the same time points. No statistically significant changes in adenosine A1 receptor or beta-actin mRNA levels were noted 48 h after the induction of ARF with HgCl2. 5. This study indicates that glycerol-induced ARF in the rat is associated with an increase in renal adenosine A1 receptor density which appears to result from increased transcription of the gene for this receptor. An increase in adenosine A1 receptor density in renal resistance vessels may explain, at least in part, the enhanced renal vasoconstrictor response to adenosine in glycerol-induced ARF that was noted in a previous study.

Decreased drug binding in uraemia: effect of indoxyl sulphate and other endogenous substances on the binding of drugs and dyes to human albumin.

Ten uraemic metabolites, alone or in combination, have been investigated by equilibrium dialysis for their effect on the binding of methyl red, methyl orange, 2-(4'-hydroxybenzeneazo)benzoic acid (HABA), phenytoin and L-tryptophan to human albumin (HSA). Indoxyl sulphate emerges as a substance likely to inhibit binding in vivo while the other metabolites were unlikely to be implicated in the binding defect of uraemic plasma. The effects of indoxyl sulphate, on the binding of HABA and methyl red, studied by equilibrium dialysis and spectroscopy respectively, indicated competitive inhibition. The results suggest that indoxyl sulphate and indole carboxylic acids may contribute to the binding defect of uraemic plasma.

Analysis of the binding of fluorescent ligands to soluble proteins. Use of simultaneous non-linear least squares regression to obtain estimates of binding parameters.

The binding of three fluorescent ligands (warfarin, dansylsarcosine and 1-anilino-8-naphthalene sulphonate) to human albumin was analysed using simultaneous non-linear least squares regression analysis. Both mock and actual fluorescence data were examined and the results indicated that reliable estimates of the binding parameters as well as the molar fluorescence of bound ligand could be obtained. The advantage of this method of analysis is that it makes full use of all the experimental data and it eliminates the need for the graphical procedures usually employed to estimate the molar fluorescence of bound ligand and its binding constants. This type of analysis can be extended to other systems where some physical property of the bound ligand varies with increasing protein concentration.

Effect of arginine on cisplatin-induced acute renal failure in the rat.

The effect of arginine on the nephrotoxicity produced by cisplatin (6.0 mg/kg i.v.) was investigated in the rat. Intravenous administration of L-arginine at doses of 0.26-2.63 g/kg at the time of cisplatin injection produced significant protection of renal function as evidenced by reductions in plasma urea and creatinine concentrations, decreased polyuria and increases in the plasma clearance of [3H]inulin and [14C]-p aminohippurate. Administration of D-arginine (2.63 g/kg i.v.) also significantly ameliorated the renal dysfunction induced by cisplatin although this protective effect was not as great as produced by the same dose of L-arginine. D-arginine, by contrast to its L-isomer, is reported to have little or no effect on renal haemodynamics. Consequently, the results of this study indicate that the protective effect of L-arginine in cisplatin nephrotoxicity involves both haemodynamic and nonhaemodynamic components.

The plasma clearance of indocyanine green in rats with acute renal failure: effect of dose and route of administration.

The pharmacokinetics of various doses (1-7.5 mg/kg i.v.) of indocyanine green (ICG) have been studied in control rats and rats with glycerol-induced acute renal failure (ARF). The pharmacokinetic changes seen at a dose of 1 mg/kg, after jugular vein administration, were significant decreases in uraemic rats in the rate of entry of ICG into the liver (k12) and in the rate of movement of dye from liver to plasma (k21). Greater and more numerous changes in pharmacokinetic parameters were recorded in experiments conducted using 4.0 and 7.5 mg/kg ICG. The results from these experiments showed that in addition to significant decreases in k12 and k21 there was a significant reduction in the rate constant for transfer of dye from liver to bile (k23). These changes were accompanied by a significant decrease in plasma clearance. In a separate series of experiments steps were taken to reduce the degree of uraemia produced by glycerol injection. The findings from these experiments showed no significant pharmacokinetic differences between control and mildly uraemic animals after administration of a dose of 7.5 mg/kg ICG. This suggests that the kinetic changes described above were a consequence of renal failure and not a direct hepato-toxic effect of glycerol.

Effect of valproic acid, its unsaturated metabolites and some structurally related fatty acids on the binding of warfarin and dansylsarcosine to human albumin.

The sites to which valproic acid and its main unsaturated metabolites (2-en-2-propyl pentanoic acid and 4-en-2-propyl pentanoic acid) bind to on human albumin were investigated by (1) measuring their ability to displace the fluorescent probes warfarin and dansylsarcosine and (2) by assessing the extent to which they inhibited the hydrolysis of 4-nitrophenyl acetate. Valproate and its metabolites displaced both warfarin and dansylsarcosine, and they also inhibited the hydrolysis of 4-nitrophenyl acetate. The order of potency for inhibition of both binding and hydrolysis was: 2-en-2-propyl pentanoic acid greater than 4-en-2-propyl pentanoic acid greater than or equal to valproate. It is concluded that valproic acid and its unsaturated metabolites can displace ligands from the warfarin binding site (site I) and the benzodiazepine/indole binding site (site II), but the primary interaction is with site II. Furthermore, the introduction of a double bond into the carbon backbone of valproate increases affinity for albumin at both sites.

A pitfall in the use of double-reciprocal plots to estimate the intrinsic molar fluorescence of ligands bound to albumin.

Double-reciprocal plots of fluorescence intensity versus protein concentration are often used to obtain the intrinsic molar fluorescence (Fb) of ligands bound to acceptor molecules such as albumin. In this paper we show that these plots develop upward concave curvature as the concentration of albumin increases. Thus linear extrapolation of such plots cannot be employed to provide accurate values of Fb. It is suggested that a direct plot of fluorescence intensity versus log protein concentration should be employed to obtain Fb.

A comparison of drug binding sites on mammalian albumins.

The fluorescent probes warfarin and dansylsarcosine are known to selectively interact with binding sites I and II, respectively, on human albumin. This paper investigates whether similar binding sites exist on bovine, dog, horse, sheep and rat albumins. Binding sites on albumins were studied by: (1) displacement of warfarin and dansylsarcosine by site I (phenylbutazone) and site II (diazepam) selective ligands; (2) the effects of non-esterified fatty acids (carbon chain lengths: C5-C20) and changes in pH (6-9) on the fluorescence of warfarin and dansylsarcosine; and (3) the ability of site selective ligands to inhibit hydrolysis of 4-nitrophenyl acetate. For bovine, dog, horse, human and sheep albumins the fluorescence of bound warfarin and dansylsarcosine was selectively decreased by phenylbutazone and diazepam, respectively. For these albumins medium chain fatty acids (C1-C12) reduced the fluorescence of dansylsarcosine (maximum inhibition with C9) whereas long chain acids (C12-C20) enhanced the fluorescence of warfarin (maximum increases with C12). In addition, changes in pH from 6 to 9 increased the fluorescence of warfarin and although site I ligands (warfarin/phenylbutazone) had no pronounced effects on 4-nitrophenyl acetate hydrolysis, site II ligands (dansylsarcosine/diazepam) significantly inhibited this reaction. Rat albumin behaved differently from the other albumins studied in that the C12-C20 fatty acids and changes in pH did not enhance the fluorescence of warfarin. Moreover, the differential effects of site I and site II ligands on the fluorescence of warfarin/dansylsarcosine and hydrolysis of 4-nitrophenyl acetate were less apparent with rat albumin. The results suggest bovine, dog, horse and sheep albumins have binding sites for warfarin and dansylsarcosine with similar properties to sites I and II on human albumin. By contrast, the warfarin binding site and to a lesser degree the dansylsarcosine site, of rat albumin have different characteristics from these sites on the other albumins studied.

Immunolocalisation of adenosine A(1) receptors in the rat kidney.

The location of adenosine A(1) receptors in the rat kidney was investigated using immunolabelling with antibodies raised to a 15-amino-acid sequence near the C-terminus of the receptor (antibody I) and to a 14-amino-acid sequence in the second extracellular loop (antibody II). In the cortex, antibody I bound to adenosine A(1) receptors in mesangial cells and afferent arterioles, whilst antibody II bound to receptors in proximal convoluted tubules. In the medulla, both antibodies bound to receptors in collecting ducts and the papillary surface epithelium. These observations provide support for the diverse functional roles previously proposed for the adenosine A(1) receptor in the kidney. The labelling of distinct but different structures in the cortex by antibodies raised to different amino acid sequences on the A(1) receptor protein suggests that differing forms of the receptor are present in this region of the kidney.

Differential expression of renal adenosine A(1) receptors induced by acute renal failure.

The distribution of renal adenosine A(1) receptors was investigated in rats with glycerol- or mercuric chloride (HgCl(2))-induced acute renal failure. Receptors were localised by autoradiography using [(3)H]8-cyclopentyl-1,3-dipropylxanthine ([(3)H]DPCPX), a selective A(1) adenosine receptor antagonist. In saline-injected control animals, significant labelling with [(3)H]DPCPX was detected in glomeruli, the inner stripe of outer medulla, and the inner medulla. Sixteen hours following induction of glycerol-induced acute renal failure (ARF), a 34% increase in labelling in glomeruli was noted compared to saline-injected controls, and by 48 hr, glomerular labelling had increased by 200%. In addition, 48 hr following glycerol injection, significant labelling was now detected in the cortical labyrinth and medullary rays whilst, in the inner medulla, labelling had decreased by 34%. By contrast to glycerol-induced ARF, the only significant change noted 48 hr following induction of HgCl(2)-induced ARF was a 39% decrease in labelling in the inner medulla. It is concluded that glycerol-induced ARF results in differential expression of renal adenosine A(1) receptors with increased expression in the cortex and reduced expression in the inner medulla. Increased density of A(1) receptors in glomeruli may account, at least in part, for the increased renal vasoconstrictor response to adenosine and depressed glomerular filtration rate noted previously in this type of acute renal failure.