Purine nucleotide cycle in rat renal cortex and medulla under conditions that mimic normal and low oxygen supply.

The distinctive feature of the renal function and metabolism implicate a possibility of excessive ATP degradation during insufficient oxygen supply. Protection of the purine ring against degradation is one among other functions of the purine nucleotide cycle (PNC). The purpose of this study was to estimate the activity of PNC in cytosol of rat renal cortex and medulla under conditions that mimic normal and low oxygen supply in vivo. In normoxic-like condition the rate of AMP deamination was 1.7 and 2.0 nmol/mg protein/min in the cytosol of cortex and medulla, respectively. Under this condition, the rate of IMP reamination was similar to that of AMP deamination. In a hypoxia-like condition the rate of AMP deamination increased by 41% in cytosol from both parts of the kidney, while the rate of IMP reamination remained unchanged in the cytosol of medulla and decreased by 46% in the cortex cytosol. Distribution of the other enzymes of the PNC, that is, adenylosuccinate synthetase and adenylosuccinate lyase, in the cytosol of cortex and medulla correlated with that observed for AMP deamination and IMP reamination potentials. At 150 microM IMP, the activity of adenylosuccinate synthetase in the cortex and medulla was 0.34 and 1.24 nmol/mg protein/min, respectively. Activity of the adenylosuccinate lyase was severalfold greater than the respective activity of the adenylosuccinate synthetase. These results show that the efficiency of PNC is about twice as high in the medulla cytosol as in the cortex cytosol, and that the activity of PNC in kidney is mainly limited by the activity of adenylosuccinate synthetase and supply of AMP.

Inhibition of cGMP-phosphodiesterase restores the glomerular effects of atrial natriuretic factor in low sodium diet rats.

It was shown that atrial natriuretic factor (ANF)-induced increase in glomerular filtration rate (GFR) is blunted in low sodium diet. We investigated whether saralasin, as an angiotensin II receptor antagonist, or verapamil, as a calcium entry blocker, or theophylline and zaprinast, as inhibitors of cGMP-phosphodiesterase activity, could restore the effect of ANF on GFR increase in low sodium diet rats. ANF alone (5 micrograms/kg bolus then 0.5 micrograms/min/kg BW maintenance) increased diuresis and natriuresis to the same extend in low and normal sodium diet rats but had no GFR-increasing effect in low sodium diet rats. Infusion of ANF in the presence of verapamil, saralasin, theophylline or zaprinast induced a significant increase in GFR and filtration fraction (FF). Administration of verapamil or saralasin alone did not alter GFR and FF whereas theophylline or zaprinast alone resulted in moderate but significant increases in both parameters. The increase of nephrogenous cGMP excretion in response to ANF infusion in low sodium diet rats was markedly lower as compared to normal sodium diet rats (243.4 +/- 43.3 vs. 444.0 +/- 35.6 pmol/min, respectively, p < 0.01). Administration of a selective inhibitor of cGMP-phosphodiesterase activity (zaprinast) abolished the differences in ANF-stimulated nephrogenous cGMP excretion in low and normal sodium diet rats. Basal and peak ANF (0.5 microM)-stimulated cGMP formation by isolated glomeruli was significantly lower in low than normal sodium diet rats (1.4 +/- 0.1 vs. 2.9 +/- 0.2 and 7.1 +/- 0.5 vs. 12.5 +/- 1.1 pmol/mg protein, for basal and ANF-stimulated cGMP formation, respectively; both p < 0.05). Zaprinast both alone and in combination with ANF, potentiated cGMP formation by glomeruli isolated from both groups of rats. In the presence of zaprinast, there were no differences in both basal and peak ANF-stimulated cGMP formation by glomeruli isolated from low and normal sodium diet rats. cGMP-phosphodiesterase activity was the same in the medulla of both groups of rats but markedly higher in the renal cortex of low sodium diet rats as compared to normal sodium diet rats (82.6 +/- 6.0 vs. 59.8 +/- 4.3, respectively; p < 0.05). These data suggest that the lack of GFR-increasing response to ANF in low sodium diet rats is primarily due to the increase of cGMP hydrolysis in glomeruli.

Adenosine deaminase restores the ability of atrial natriuretic peptide to induce glomerular hyperfiltration in low-sodium rats.

Administration of a large dose of atrial natriuretic peptide is associated with an increase in glomerular filtration rate, diuresis and natriuresis in normal-sodium rats. However, glomerular hyperfiltration induced by atrial natriuretic peptide is markedly decreased in low-sodium rats. Glomerular insensitivity to atrial natriuretic peptide may be due to increased activity of the renin-angiotensin system in low-sodium rats and to an accompanying hypersensitivity to adenosine. The results indicate that attenuated glomerular responses to atrial natriuretic peptide are restored by the administration of adenosine deaminase in low-sodium rats. Moreover atrial natriuretic peptide markedly increases the urinary excretion of adenosine deaminase, which may be due to increased permeability of glomeruli to the enzyme.

The distribution of enzymes involved in purine metabolism in rat kidney.

Adenosine produced from 5'-AMP has been proposed as a mediator of intrinsic renal regulation. The rates of 5'-AMP and adenosine metabolism are dependent on the activities of enzyme involved in purine metabolism. The activities of adenosine kinase (AK), adenosine deaminase (ADA), 5'-nucleotidase (5'-NT), AMP deaminase, xanthine oxidase and purine nucleoside phosphorylase were measured in cytosolic and membrane fractions from glomeruli, cortical tubules, medullary thick ascending limb of Henle (MTAL) and collecting duct prepared from rat kidney by combinations of sieving and sucrose density gradient centrifugation techniques. In the cytoplasm of glomeruli cells, the activity ratios of ADA/AK and AMP deaminase/5'-NT were 70 and 2.4, respectively. The highest activity of 5'-NT was found in membrane fractions of cortical tubules where it was equally distributed between luminal and antiluminal membranes. Membrane fractions of MTAL did not contain detectable amounts of adenosine deaminase activity. The highest activity of xanthine oxidase and purine nucleoside phosphorylase was in the cytoplasm fraction of glomeruli. These results suggest that deamination of AMP and adenosine may be favored in the cytoplasm of glomeruli cells. In contrast, in the extracellular space of glomeruli and especially in the cortical tubule, AMP can be converted preferentially to adenosine by 5'-NT.

The purine nucleotide cycle activity in renal cortex and medulla.

Formation of adenine nucleotides, IMP, malate + fumarate, ammonia, adenosine, and inosine + hypoxanthine + uric acid were measured in cytosolic extracts from renal cortex and medulla. The order of substrate addition was IMP, then 2-deoxyglucose, then P-creatine. Compared with cortex, medulla showed greater rates of formation of adenosine triphosphate (ATP) from P-creatine, of adenosine monophosphate (AMP) from 2-deoxyglucose, and of total adenine nucleotides from IMP. These results suggest that the purine nucleotide cycle is more active in medulla than in cortex. This cycle may provide a mechanism in medulla for storing purine nucleotides which can be used to restore ATP pools in the relatively hypoxic conditions of this part of the kidney.