M. S. Yates

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 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.

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

1 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. 2 Rats treated with 8‐PT i.v. had significantly lower plasma urea and creatinine levels at 24 and 48 h compared to untreated animals. 3 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. 4 [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. 5 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. 6 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. 7 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. 8 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 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 mg kg−1 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 0.3 mg kg−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.;

Immunolocalisation of adenosine A1 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.

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.

Alternative learning environments: what do they contribute to professional development of medical students?

Special study modules provide opportunities for students to develop lifelong learning skills and develop areas of interest. The GMC also recommends opportunities to study topics not included in the core medical curriculum. This paper reports the evaluation of modules based within alternative learning environments developed to provide students with experiences outside traditional medical, scientific or academic cultures. The attachment was highly rated as contributing to professional development, provided novel learning experiences, and was stimulating for both students and supervisors. All students achieved new skills that they felt were pertinent to their personal development. Assessment was by standardized pro forma, including generic transferable skills and module specific outcomes. Whilst overall assessment grades were comparable to similar course components, unease amongst both students and supervisors was expressed reflecting anxieties in the diversity of workloads and assessment. Attempts to standardize assessment across the diversity of modules did not reduce anxieties and potentially detracted from the learning experiences.

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.

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

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. 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‐ethylcarboxamido adenosine ([3H]‐CGS 21680), a selective adenosine A2a agonist. 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. 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 β‐actin at the same time points. No statistically significant changes in adenosine A1 receptor or β‐actin mRNA levels were noted 48 h after the induction of ARF with HgCl2. 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.

The Protective Effect of Glycine in Cisplatin Nephrotoxicity: Inhibition with NG-Nitro-l-arginine Methyl Ester

Abstract— The effect of glycine on the acute changes in renal haemodynamics and nephrotoxicity produced by cisplatin was investigated in the rat. Cisplatin (6·0 mg kg−1, i.v.) injection in anaesthetized rats produced, over a period of 2 h, falls of approximately 50% in renal blood flow (RBF) and the clearance of [3H]inulin (CLIN), effects which were prevented by co‐administration of glycine (1·0 g kg−1). Infusion of the nitric oxide (NO) synthase‐inhibitor NG‐nitro‐l‐arginine methyl ester, l‐NAME (10 μg min−1 kg−1, i.v.), abolished glycines ability to maintain RBF in cisplatin‐injected rats whilst partially inhibiting the ability of glycine to preserve CLIN. Treatment of cisplatin‐injected rats with glycine (1·0 g kg−1, i.v.) significantly ameliorated the nephrotoxic effects of cisplatin (6·0 mg kg−1) as judged by improvements in a range of indices of renal function which included plasma urea and creatinine concentrations, urine output, sodium excretion, CLIN and the clearance of [14C]p‐aminohippurate. Administration of l‐NAME (1·0 mg kg−1, i.v.) to rats which received cisplatin and glycine significantly inhibited the reno‐protective effect of glycine. However, l‐NAME administration to rats which were treated only with cisplatin did not result in any potentiation of cisplatin nephrotoxicity. The findings of this study suggest that glycine can block the acute falls in RBF and CIN produced by cisplatin by a mechanism which involves the production of NO. Furthermore, the results indicate that these renal haemodynamic actions of glycine are responsible, at least in part, for the ability of this amino acid to ameliorate cisplatin nephrotoxicity.