Vol.
170,
No.
July
31, 1990
2, 1990
ATPENUATED
BIOCHEMICAL
COMMUNICATIONS
RESEARCH
GLONERULARRESPONSES TOATIUAL NATRIURETIC LOW-SODIUM RATS IS PREVENTED BY TREOPHYLLINE
Department
June
BIOPHYSICAL
Pages
S. Angielski,
Received
AND
6,
G. Kuchta, M. Redlak, of
Clinical
596-602
FACTOR
IN
and M. Szczepanska-Konkel
Biochemistry, Gdansk,Poland
Medical
Academy,
1990
Atria1 natriuretic factor administered in the large dose did not change glomerular filtration rate, but it was diuretic in In response to ANF, excretion low-sodium rats. of c-GMP was decreased in low-sodium rats in comparison with normal-sodium rats. Theophylline prevented glomerular response to ANF. ANFstimulated c-GMP accumulation in isolated glomeruli was more diminished in lowthan normal sodium rats. results These indicate that attenuated glomerular responses to ANF in lowsodium rats might be due to increase of plasm$+Angiotensin II (Ang II) level, which increases intracellular Ca concentration. Theophy$l+ine can potentiate the renal response to ANF. We suggest that Ca -activated c-GMP phosphodiesterase plays a major role in the regulation of intracellular accumulation of c-GMP in glomeruli exposed to ANF. 01990 Academic mess, 1°C. Administration of ANF is associated with increases in GFR and ANF may act on the glomerulus , at least in natriuresis (1,2,3). part, by interaction with other hormonal systems such as Ang II (5) or local factors such as adenosine (6) or (41, vasopresin Intrarenal infusion of Ang II attenuated the prostaglandins (7). glomerular hyperfiltration induced by ANF (4), whereas vasopresin potentiated the natriuretic effect of ANF without altering GFR (5). The decrease of intrarenal adenosine concentration caused by infusion of adenosine deaminase markedly potentiated the hyperfiltration response to ANF (6). Blunted hyperfiltration (8,9) and reduced natriuretic response to ANF have been reported in low-sodium diet in humans. It has been shown, that ANF receptor density in glomeruli was severalfold greater in lowsodium rats than in high-sodium rats. However, ANF-dependent cGMP generation, in the presence of an inhibitor of the cyclic Abbreviations: ANF, atria1 natriuretic factor; Ang II, angiotensin II; cw, guanosine 3,5-cyclic monophosphate; GFR, glomerular filtration rate; IBXX, isobutyl methylxanthine. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
596
Vol.
170, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phosphodiesterase, did not differ between glomeruli from rats on low- compared with high-sodium diet (10). Therefore, the decrease in ANF-provoked cGMP accumulation observed in the glomeruli with increased receptor density may be due to activation of phosphodiesterase or alternatively inhibition of guanylate cyclase. We therefore attempted to define the role of cyclic nucleotide phosphodiesterase in blunted glomerular responses to ANF in low-sodium rats. This was done by evaluating the effects of ANF on GFR and urinary cGMP excretion before and after the inhibition of nucleotide phosphodiesterase with theophylline. Moreover, we measured ANF-dependent cGMP generation in glomeruli isolated from rats on low and normal sodium diets.
MATERIALS
ANDMECHODS
Clearances experiments were performed on male Wistar rats (weight 200-250g). Animals were divided into two groups of 10 rats and given either the low-sodium diet (0.005 mm01 Na+/g) or normal-sodium diet. The low sodium rats were given distilled water to drink. After 6 days on the diets the rats were anasthesized with phenobarbital (50 mg/kg body weight). A catheter was placed in the femoral artery for administration of inulin, paraaminohippurate or drugs dissolved in isotonic saline. The urinary bladder was cannulated for urine collection. A 60 min equilibration period occured before experiments were started. The saline was infused at O.O30ml/min. ANF was infused at 1 ug/min/kg body weight for 20 min. Two clearences of 10 min each were obtained. When theophylline was tested, the first two 10 min baseline clearence collection was obtained followed by infusion of theo:phylline (0.2 umol/min/kg body weight) and two more clearences measurements. Then ANF was infused in the same dose as previously and additional two 10 min clearences periods were obtained. Preparation of glomeruli. Following the decapitation of rats, the kidneys were perfused with phosphate-buffered saline (PBS) containing 5 mM glucose. After perfusion, kidneys were removed, sliced and squeezed through sieve with a pore size of 250 um and then rinsed. The suspension was buttered through a 120 u.m sieve and centrifuged at 1OOg for 90 sec. The supernatant was discarded and the pellet was resuspended. Glomeruli retained on the 75 um sieve were collected in a centrifuge tube and washed three times twice Hank's by centrifugation in PBS and once in ice-cold The purity of each preparation was balanced salt solution (HBS). evaluated by light microscopy. Glomeruli were suspended in Measurement of c-GMP in glOmerUli. Hanks solution (in mM: NaCl 110; Na2HP04 2.5; KC1 5.0; MgS04 1.0; sodium succinate 5.0;) glucose 5.0; HC03 25; CaCl 1.0; Incu 2 ation mixture was equilibrated with 95% 02 and 5% C02. Incubation temp. was 37O. The cGMP content was analysed using c-GMP RIA kit (Amersham). described Adenosine was determined radioimmunologicaly as previously (13). Inulin was measured according to (11) and
597
Vol.
170,
No.
2, 1990
BIOCHEMICAL
paraaminohippurate according determined according to gravimetrically and sodium Synthetic ANF (rat ANF 8-33) (Belmont CA). 14-C Adenosine land).
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
to (12). Glomerular protein was Urine volume was measured (14). concentration by flame photometer. was from Peninsula Laboratories was purchased from Amersham (Eng-
RESULTS
In normal sodium rats, infusion of ANF resulted in an increase of urine flow, urinary sodium excretion and GFR (Table 1) which was accompanied by significant increments in urinary adenosine adenosine levels did not increase excrection, whereas plasma Infusion of ANF elicited marked increase in significantly. cGMP increased nephrogenic cGMP excretion (Fig. 1). Also plasma from 2.1kO.l to 3.920.7 during ANF infusion. Renal plasma flow did not change during infusion of ANF. GFR did not change in response to infusion In low-sodium rats, flow and urinary sodium increased (Table 2). of ANF. Urine during ANF nephrogenic cGMP excretion increased, Although the increase (65+12 vs 115215) was markedly less than infusion, in normal-sodium rats. Baseline plasma cGMP concentrations were 1.6kO.l and 2.120.3 for low- and normal-salt rats, respectively (p
170,
No.
July
31, 1990
2, 1990
ATPENUATED
BIOCHEMICAL
COMMUNICATIONS
RESEARCH
GLONERULARRESPONSES TOATIUAL NATRIURETIC LOW-SODIUM RATS IS PREVENTED BY TREOPHYLLINE
Department
June
BIOPHYSICAL
Pages
S. Angielski,
Received
AND
6,
G. Kuchta, M. Redlak, of
Clinical
596-602
FACTOR
IN
and M. Szczepanska-Konkel
Biochemistry, Gdansk,Poland
Medical
Academy,
1990
Atria1 natriuretic factor administered in the large dose did not change glomerular filtration rate, but it was diuretic in In response to ANF, excretion low-sodium rats. of c-GMP was decreased in low-sodium rats in comparison with normal-sodium rats. Theophylline prevented glomerular response to ANF. ANFstimulated c-GMP accumulation in isolated glomeruli was more diminished in lowthan normal sodium rats. results These indicate that attenuated glomerular responses to ANF in lowsodium rats might be due to increase of plasm$+Angiotensin II (Ang II) level, which increases intracellular Ca concentration. Theophy$l+ine can potentiate the renal response to ANF. We suggest that Ca -activated c-GMP phosphodiesterase plays a major role in the regulation of intracellular accumulation of c-GMP in glomeruli exposed to ANF. 01990 Academic mess, 1°C. Administration of ANF is associated with increases in GFR and ANF may act on the glomerulus , at least in natriuresis (1,2,3). part, by interaction with other hormonal systems such as Ang II (5) or local factors such as adenosine (6) or (41, vasopresin Intrarenal infusion of Ang II attenuated the prostaglandins (7). glomerular hyperfiltration induced by ANF (4), whereas vasopresin potentiated the natriuretic effect of ANF without altering GFR (5). The decrease of intrarenal adenosine concentration caused by infusion of adenosine deaminase markedly potentiated the hyperfiltration response to ANF (6). Blunted hyperfiltration (8,9) and reduced natriuretic response to ANF have been reported in low-sodium diet in humans. It has been shown, that ANF receptor density in glomeruli was severalfold greater in lowsodium rats than in high-sodium rats. However, ANF-dependent cGMP generation, in the presence of an inhibitor of the cyclic Abbreviations: ANF, atria1 natriuretic factor; Ang II, angiotensin II; cw, guanosine 3,5-cyclic monophosphate; GFR, glomerular filtration rate; IBXX, isobutyl methylxanthine. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
596
Vol.
170, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phosphodiesterase, did not differ between glomeruli from rats on low- compared with high-sodium diet (10). Therefore, the decrease in ANF-provoked cGMP accumulation observed in the glomeruli with increased receptor density may be due to activation of phosphodiesterase or alternatively inhibition of guanylate cyclase. We therefore attempted to define the role of cyclic nucleotide phosphodiesterase in blunted glomerular responses to ANF in low-sodium rats. This was done by evaluating the effects of ANF on GFR and urinary cGMP excretion before and after the inhibition of nucleotide phosphodiesterase with theophylline. Moreover, we measured ANF-dependent cGMP generation in glomeruli isolated from rats on low and normal sodium diets.
MATERIALS
ANDMECHODS
Clearances experiments were performed on male Wistar rats (weight 200-250g). Animals were divided into two groups of 10 rats and given either the low-sodium diet (0.005 mm01 Na+/g) or normal-sodium diet. The low sodium rats were given distilled water to drink. After 6 days on the diets the rats were anasthesized with phenobarbital (50 mg/kg body weight). A catheter was placed in the femoral artery for administration of inulin, paraaminohippurate or drugs dissolved in isotonic saline. The urinary bladder was cannulated for urine collection. A 60 min equilibration period occured before experiments were started. The saline was infused at O.O30ml/min. ANF was infused at 1 ug/min/kg body weight for 20 min. Two clearences of 10 min each were obtained. When theophylline was tested, the first two 10 min baseline clearence collection was obtained followed by infusion of theo:phylline (0.2 umol/min/kg body weight) and two more clearences measurements. Then ANF was infused in the same dose as previously and additional two 10 min clearences periods were obtained. Preparation of glomeruli. Following the decapitation of rats, the kidneys were perfused with phosphate-buffered saline (PBS) containing 5 mM glucose. After perfusion, kidneys were removed, sliced and squeezed through sieve with a pore size of 250 um and then rinsed. The suspension was buttered through a 120 u.m sieve and centrifuged at 1OOg for 90 sec. The supernatant was discarded and the pellet was resuspended. Glomeruli retained on the 75 um sieve were collected in a centrifuge tube and washed three times twice Hank's by centrifugation in PBS and once in ice-cold The purity of each preparation was balanced salt solution (HBS). evaluated by light microscopy. Glomeruli were suspended in Measurement of c-GMP in glOmerUli. Hanks solution (in mM: NaCl 110; Na2HP04 2.5; KC1 5.0; MgS04 1.0; sodium succinate 5.0;) glucose 5.0; HC03 25; CaCl 1.0; Incu 2 ation mixture was equilibrated with 95% 02 and 5% C02. Incubation temp. was 37O. The cGMP content was analysed using c-GMP RIA kit (Amersham). described Adenosine was determined radioimmunologicaly as previously (13). Inulin was measured according to (11) and
597
Vol.
170,
No.
2, 1990
BIOCHEMICAL
paraaminohippurate according determined according to gravimetrically and sodium Synthetic ANF (rat ANF 8-33) (Belmont CA). 14-C Adenosine land).
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
to (12). Glomerular protein was Urine volume was measured (14). concentration by flame photometer. was from Peninsula Laboratories was purchased from Amersham (Eng-
RESULTS
In normal sodium rats, infusion of ANF resulted in an increase of urine flow, urinary sodium excretion and GFR (Table 1) which was accompanied by significant increments in urinary adenosine adenosine levels did not increase excrection, whereas plasma Infusion of ANF elicited marked increase in significantly. cGMP increased nephrogenic cGMP excretion (Fig. 1). Also plasma from 2.1kO.l to 3.920.7 during ANF infusion. Renal plasma flow did not change during infusion of ANF. GFR did not change in response to infusion In low-sodium rats, flow and urinary sodium increased (Table 2). of ANF. Urine during ANF nephrogenic cGMP excretion increased, Although the increase (65+12 vs 115215) was markedly less than infusion, in normal-sodium rats. Baseline plasma cGMP concentrations were 1.6kO.l and 2.120.3 for low- and normal-salt rats, respectively (p
