Br. J. Pharmacol. (1992), 105, 869-874
'."
Effect of x.-human atrial natriuretic peptide dopamine in the rat kidney
on
Macmillan Press Ltd, 1992
the synthesis of
'P. Soares-da-Silva & 2M. Helena Fernandes Department of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal 1 The present study has examined the influence of ac-human atrial natriuretic peptide (ac-hANP) on the synthesis of dopamine and its deamination into 3,4-dihydroxyphenylacetic acid (DOPAC) in rat kidney slices loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). 2 ax-hANP (3.3 and 330 nM) was found to produce a marked reduction (63- 78% reduction) in the time-dependent accumulation of newly-formed dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with 10 ytM L-DOPA. oc-hANP (330 nM) was also found to decrease the accumulation of newly-formed dopamine (45- 66% reduction) and DOPAC (38- 61% reduction) in experiments in which increasing concentrations (1- 100 t1M) of L-DOPA were used. This inhibitory effect was found to be potentiated by zaprinast (M&B 22,948; 10IM), a guanosine cyclic 3',5'-monophosphate (cyclic GMP) phosphodiesterase inhibitor. Alone, zaprinast also decreased the accumulation of both dopamine (54-71% reduction) and DOPAC (73-92% reduction). 3 In kidney homogenates, a-hANP (330 nM) was found to affect neither the formation of dopamine nor its deamination to DOPAC. 4 Both ac-hANP (330 nM) and zaprinast (10 tiM) were found not to affect the formation of dopamine and DOPAC in kidney slices obtained from rats on a high salt diet during the previous 6 weeks. A similar situation was also found to occur when kidney slices obtained from 24-months old rats were used. 5 The results obtained suggest that the inhibitory effect of ac-hANP on the renal synthesis of dopamine is dependent on the activation of a membrane-operated mechanism, coupled to the enzyme guanylate cyclase, controlling the entry of L-DOPA into the cells. Keywords: ac-Human atrial natriuretic peptide; kidney; dopamine; zaprinast; high salt diet; aging
Introduction Atrial natriuretic peptides (ANPs) exert profound effects on renal function and play an important role in the regulation of sodium balance and central blood volume. Although a full expression of the natriuretic effects of ANPs is thought to depend predominantly on their renal haemodynamic actions, they may also directly alter tubular sodium reabsorption through the activation of specific receptors (Salazar et al., 1986; Sonneberg et al., 1986). However, several studies performed in the rat have shown that the natriuretic response to ANPs was completely blocked by pretreatment with dopamine receptor antagonists; the most effective compounds were those blocking DI-receptors (for review see Murphy & Bass, 1990). Since some of the renal effects of ANPs, namely those concerning vasodilatation and inhibition of tubular sodium reabsorption are also shared by the catecholamine dopamine, it has been suggested that the renal effects of ANPs might be mediated through enhanced renal dopaminergic activity. Conversely, other authors have demonstrated, in dogs and man, that the natriuretic effect of cc-human ANP (oc-hANP) did not depend on a permissive role of dopamine (Murphy et al., 1988; Freestone et al., 1989; Lewis et al., 1989). In the kidney, ANPs activate specific receptors coupled to the particulate form of guanylate cyclase, the activation of which results in an increase in the generation of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) (Kim et al., 1989). By contrast, type DI-receptors in renal vascular smooth muscle cells and tubular epithelial cells are coupled to the enzyme adenylate cyclase the activation of which is accom-
I Author for correspondence. 2 Permanent address: Faculty of Medical Dentistry, 4200 Porto, Portugal.
panied by an increase in the formation of adenosine 3',5'cyclic monophosphate (cyclic AMP) (for review see Felder et al., 1989). Since natriuresis induced by the infusion of cchANP closely parallels the increase in the urinary excretion of cyclic GMP (Lewis et al., 1988), one possible explanation for the permissive role of dopamine on the natriuretic effects of ANPs, as has been found to occur in the rat, might be related to the possibility that peptide-induced natriuresis, may be dependent on the mobilization of dopamine of renal origin. In fact, renal tissues are endowed with an enormous capacity to synthesize dopamine; tubular epithelial cells, namely those of proximal convoluted tubules, are endowed with a high aromatic L-amino acid decarboxylase (AAAD) activity and filtered dihydroxyphenylalanine (DOPA) is converted to dopamine after being taken up into this cellular compartment (Baines & Chan, 1980). Considering that endogenous intrarenal dopamine may act physiologically in the control of the renal excretion of sodium, as a result of activation of DI-receptors located in tubular epithelial cells (Siragy et al., 1989), the present study was undertaken to examine the effects of ax-hANP on the synthesis of dopamine in rat renal tissues loaded with exogenous L-DOPA. A preliminary account of these findings was reported initially at the 3rd International Conference on Peripheral Dopamine (Soares-da-Silva & Fernandes, 1990a).
Methods Male Wistar rats (Bioterio do Instituto Gulbenkian de Ciencia, Oeiras, Portugal) aged 45-60 days and weighing 200-280 g were used in the experiments. Animals were kept two per cage under controlled environmental conditions (12 h light/dark cycle and room temperature, 24°C). Food and tap
870
P. SOARES-DI-SILVA & M.H. FERNANDES
water were allowed ad libitum. The experiments were all carried out during day time. The rats were killed by decapitation under ether anaesthesia and both kidneys were removed and rinsed free from blood with saline (0.9% NaCl). The kidneys were placed on an ice cold glass plate, the kidney poles removed and renal slices approximately 1.5 mm thick, containing both the cortex and the medulla, and weighing about 90 mg wet weight were prepared with a scalpel. Thereafter, renal slices were preincubated during 60 min in warm (370C) and gassed (95% 02 and 5% CO2) 10 ml Krebs solution. The Krebs solution had the following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.4, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, EDTA 0.4, ascorbic acid 0.57 and glucose 11; (-)-a-methyl-p-tyrosine (50 JM) and copper sulphate (1O gM) were also added to the Krebs solution to inhibit the enzyme tyrosine hydroxylase and the endogenous inhibitors of dopamine P-hydroxylase, respectively. After preincubation, renal slices were incubated for 5, 10, 20 or 30 min in gassed and warm Krebs solution with added L-DOPA (10 4M). In another set of experiments, kidney slices were incubated for 15 min with increasing concentrations of L-DOPA (1-100 ILM) added to the medium. In experiments in which the effects of a-hANP (3.3 and 330 nM) and zaprinast (M&B 22,948; 10 PM), a relatively specific guanosine cyclic GMP phosphodiesterase inhibitor, were tested, the compounds were present during the preincubation and incubation periods. The preincubation and incubation were carried out in a shaking water bath at 370C, in an atmosphere of 95% 02 and 5% CO2; renal slices were incubated individually in glass vials containing 10 ml Krebs solution. After the incubation, renal slices were collected, washed for 30 min in ice cold Krebs solution, blotted with filter paper, minced with fine scissors and placed in 2 ml of 0.2 M perchloric acid at 40C for the next 24 h, before quantification of tissue catecholamines. Some experiments with kidney slices were performed with tissues taken from animals on a high salt diet (1% sodium chloride in the drinking fluid) during the previous 6 weeks. The daily sodium intake of rats on the high salt and normal salt diet averaged, respectively, 0.5 and 5 mmol 100 g-' body weight. In another series of experiments, kidney slices were obtained from 24-months old rats (considered to be old). In another set of experiments, kidney homogenates, instead of tissue slices, were used. Whole kidneys were homogenized in a modified Krebs solution with Duall-Kontes homogenizers and kept continuously on ice. The modified Krebs solution consisted of a medium similar to that described above with the exception that NaCl was reduced to 50 mM; the osmolarity of the medium was kept constant by the addition of 68 mM choline chloride. Aliquots of 1.0 ml of kidney homogenates plus 1.5 ml Krebs solution were placed in glass test tubes incubated for 60 min; thereafter, L-DOPA (0. 1 - 10.0 jM) was added to the medium for a further 15 min. In experiments in which the effect of x-hANP (330 nM) was tested, the peptide was present during the preincubation and incubation periods. During preincubation and incubation, kidney homogenates were continuously shaken and gassed (95% 02 and 5% CO2) at a constant temperature of 37TC. The reaction was stopped by adding 250 Al of 2 M perchloric acid and the preparations placed at 40C for 60 min; thereafter, kidney homogenates were centrifuged (2000 r.p.m., 2 min, 4°C) and aliquots of 1.5 ml of the supernatant used for the assay of L-DOPA, dopamine and DOPAC. The assay of L-DOPA, dopamine, noradrenaline and DOPAC in renal tissues and kidney homogenates was performed by means of high performance liquid chromatography with electrochemical detection, as previously described (Soares-da-Silva & Fernandes, 1991a). In brief, aliquots of 1.5 ml perchloric acid in which the tissues had been kept or 1.5 ml of supernatant of kidney homogenates were placed in 5 ml conical-based glass vials with 50 mg alumina and the pH of the samples immediately adjusted to pH 8.6 by the addition of Tris buffer. The adsorbed cate-
cholamines were then eluted from the alumina with 200 jl of 0.2 M perchloric acid on Millipore microfilters (MF 1); 50 gl of the eluate was injected into a high performance liquid chromatograph (Gilson Medical Electronics, Villiers le Bel, France). The lower limits for detection of L-DOPA, dopamine, noradrenaline and DOPAC were 1.0, 1.4, 0.9 and 2.5 pmol g- ', respectively. The protein content of the homogenates (mg of protein per g of tissue) was determined by the method of Lowry et al. (1951), with human serum albumin as a standard. Mean values ± s.e.mean of n experiments are given. Significance of differences between two means was estimated by Student's t test for unpaired data. Significance of difference between one control and several experimental groups was evaluated by the Tuckey-Kramer method (Sokal & Rohlf, 1981). A P value less than 0.05 was assumed to denote a significant difference. 3,4-Dihydroxyphenylacetic acid (DOPAC), L-dihydroxyphenylalanine (L-DOPA), dopamine hydrochloride, a-human atrial natriuretic peptide, (-)-a-methyl-p-tyrosine and noradrenaline bitartrate were purchased from Sigma Chemical Comany (St. Louis, Mo, U.S.A.) and zaprinast (M&B 22,948; 2-o-propoxyphenyl-8-azapurin-6-one) was kindly donated by the manufacturer (May & Backer, Ltd, Dagenham, Essex).
Results Incubation of renal tissues with 10 AM L-DOPA for 5, 10, 20 and 30 min or with increasing concentrations of L-DOPA for 15 min resulted, respectively, in a time- (Figure 1) and concentration-dependent (Figure 2) accumulation of newlyformed dopamine; the amount of the amine accumulated in the tissue reached values as high as 11 nmol g'- when 100 jAM L-DOPA was added to the medium (Figure 2). The accumulation of DOPAC, the deaminated metabolite of dopamine, in kidney slices was also dependent on the incubation time (Figure 1) and on the concentration of L-DOPA used (Figure 2) reaching values as high as 32 nmol g' when 100 AM LDOPA was used (Figure 2). In kidney slices incubated in the absence of exogenous L-DOPA, the dopamine and noradrenaline tissue concentrations were 0.018 ± 0.004 and 0.709 ± 0.041 nmol g-' (n = 8), respectively; DOPAC was not detectable under these experimental conditions. The tissue levels of noradrenaline in kidney slices did not significantly change in the course of these experiments even when 100 AM L-DOPA was used (data not shown, but see Fernandes & Soares-da-Silva, 1990). Incubation of kidney homogenates with increasing concentrations of L-DOPA (0.1-10.0 1M) for 15 min resulted in a concentrationdependent accumulation of newly-formed dopamine (Figure 3); the formation of DOPAC was dependent on the concentration of L-DOPA added to the medium (Figure 3). The addition of a-hANP (330 nM) to the incubation medium resulted in a marked decrease (63-78% reduction) in the time-dependent accumulation of dopamine and DOPAC in kidney slices incubated with 10 jM L-DOPA (Figure 1). The inhibitory effect of the peptide on the accumulation of newly-formed dopamine and DOPAC was also observed when 3.3 nM a-hANP was used; this only achieved statistical significance at 20 and 30 min of incubation (Figure 1). As shown in Figure 2, a-hANP (330 nM) was also found to decrease the accumulation of newly-formed dopamine in experiments in which increasing concentrations of L-DOPA were added to the medium during the 15 min incubation period. Under these experimental conditions, the inhibitory effect of a-hANP (45-66% reduction) could be observed at all concentrations of L-DOPA, even at 100 gM L-DOPA (Figure 2). The inhibitory effect of a-hANP on the accumulation of newly-formed dopamine was also accompanied by a reduction in the formation of DOPAC; this effect was similar (38-61% reduction) to that observed for
RENAL DOPAMINE SYNTHESIS AND ac-hANP
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L-DOPA (pM) Figure 2 Effect of a-human atrial natriuretic peptide (oc-hANP; 330 nM), zaprinast (10 pM) and x-hANP plus zaprinast on the accumulation of (a) dopamine and (b) 3,4-dihydroxyphenylacetic acid (DOPAC) (in nmol g-') in rat kidney slices incubated with increasing concentrations of exogenous L-DOPA (1-1I00 M) for 15 min. Each point represents the mean of five to six experiments per group; vertical lines show s.e.mean. Significantly different from corresponding values of control using the Tuckey Kramer method (*P
'."
Effect of x.-human atrial natriuretic peptide dopamine in the rat kidney
on
Macmillan Press Ltd, 1992
the synthesis of
'P. Soares-da-Silva & 2M. Helena Fernandes Department of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal 1 The present study has examined the influence of ac-human atrial natriuretic peptide (ac-hANP) on the synthesis of dopamine and its deamination into 3,4-dihydroxyphenylacetic acid (DOPAC) in rat kidney slices loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). 2 ax-hANP (3.3 and 330 nM) was found to produce a marked reduction (63- 78% reduction) in the time-dependent accumulation of newly-formed dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with 10 ytM L-DOPA. oc-hANP (330 nM) was also found to decrease the accumulation of newly-formed dopamine (45- 66% reduction) and DOPAC (38- 61% reduction) in experiments in which increasing concentrations (1- 100 t1M) of L-DOPA were used. This inhibitory effect was found to be potentiated by zaprinast (M&B 22,948; 10IM), a guanosine cyclic 3',5'-monophosphate (cyclic GMP) phosphodiesterase inhibitor. Alone, zaprinast also decreased the accumulation of both dopamine (54-71% reduction) and DOPAC (73-92% reduction). 3 In kidney homogenates, a-hANP (330 nM) was found to affect neither the formation of dopamine nor its deamination to DOPAC. 4 Both ac-hANP (330 nM) and zaprinast (10 tiM) were found not to affect the formation of dopamine and DOPAC in kidney slices obtained from rats on a high salt diet during the previous 6 weeks. A similar situation was also found to occur when kidney slices obtained from 24-months old rats were used. 5 The results obtained suggest that the inhibitory effect of ac-hANP on the renal synthesis of dopamine is dependent on the activation of a membrane-operated mechanism, coupled to the enzyme guanylate cyclase, controlling the entry of L-DOPA into the cells. Keywords: ac-Human atrial natriuretic peptide; kidney; dopamine; zaprinast; high salt diet; aging
Introduction Atrial natriuretic peptides (ANPs) exert profound effects on renal function and play an important role in the regulation of sodium balance and central blood volume. Although a full expression of the natriuretic effects of ANPs is thought to depend predominantly on their renal haemodynamic actions, they may also directly alter tubular sodium reabsorption through the activation of specific receptors (Salazar et al., 1986; Sonneberg et al., 1986). However, several studies performed in the rat have shown that the natriuretic response to ANPs was completely blocked by pretreatment with dopamine receptor antagonists; the most effective compounds were those blocking DI-receptors (for review see Murphy & Bass, 1990). Since some of the renal effects of ANPs, namely those concerning vasodilatation and inhibition of tubular sodium reabsorption are also shared by the catecholamine dopamine, it has been suggested that the renal effects of ANPs might be mediated through enhanced renal dopaminergic activity. Conversely, other authors have demonstrated, in dogs and man, that the natriuretic effect of cc-human ANP (oc-hANP) did not depend on a permissive role of dopamine (Murphy et al., 1988; Freestone et al., 1989; Lewis et al., 1989). In the kidney, ANPs activate specific receptors coupled to the particulate form of guanylate cyclase, the activation of which results in an increase in the generation of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) (Kim et al., 1989). By contrast, type DI-receptors in renal vascular smooth muscle cells and tubular epithelial cells are coupled to the enzyme adenylate cyclase the activation of which is accom-
I Author for correspondence. 2 Permanent address: Faculty of Medical Dentistry, 4200 Porto, Portugal.
panied by an increase in the formation of adenosine 3',5'cyclic monophosphate (cyclic AMP) (for review see Felder et al., 1989). Since natriuresis induced by the infusion of cchANP closely parallels the increase in the urinary excretion of cyclic GMP (Lewis et al., 1988), one possible explanation for the permissive role of dopamine on the natriuretic effects of ANPs, as has been found to occur in the rat, might be related to the possibility that peptide-induced natriuresis, may be dependent on the mobilization of dopamine of renal origin. In fact, renal tissues are endowed with an enormous capacity to synthesize dopamine; tubular epithelial cells, namely those of proximal convoluted tubules, are endowed with a high aromatic L-amino acid decarboxylase (AAAD) activity and filtered dihydroxyphenylalanine (DOPA) is converted to dopamine after being taken up into this cellular compartment (Baines & Chan, 1980). Considering that endogenous intrarenal dopamine may act physiologically in the control of the renal excretion of sodium, as a result of activation of DI-receptors located in tubular epithelial cells (Siragy et al., 1989), the present study was undertaken to examine the effects of ax-hANP on the synthesis of dopamine in rat renal tissues loaded with exogenous L-DOPA. A preliminary account of these findings was reported initially at the 3rd International Conference on Peripheral Dopamine (Soares-da-Silva & Fernandes, 1990a).
Methods Male Wistar rats (Bioterio do Instituto Gulbenkian de Ciencia, Oeiras, Portugal) aged 45-60 days and weighing 200-280 g were used in the experiments. Animals were kept two per cage under controlled environmental conditions (12 h light/dark cycle and room temperature, 24°C). Food and tap
870
P. SOARES-DI-SILVA & M.H. FERNANDES
water were allowed ad libitum. The experiments were all carried out during day time. The rats were killed by decapitation under ether anaesthesia and both kidneys were removed and rinsed free from blood with saline (0.9% NaCl). The kidneys were placed on an ice cold glass plate, the kidney poles removed and renal slices approximately 1.5 mm thick, containing both the cortex and the medulla, and weighing about 90 mg wet weight were prepared with a scalpel. Thereafter, renal slices were preincubated during 60 min in warm (370C) and gassed (95% 02 and 5% CO2) 10 ml Krebs solution. The Krebs solution had the following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.4, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, EDTA 0.4, ascorbic acid 0.57 and glucose 11; (-)-a-methyl-p-tyrosine (50 JM) and copper sulphate (1O gM) were also added to the Krebs solution to inhibit the enzyme tyrosine hydroxylase and the endogenous inhibitors of dopamine P-hydroxylase, respectively. After preincubation, renal slices were incubated for 5, 10, 20 or 30 min in gassed and warm Krebs solution with added L-DOPA (10 4M). In another set of experiments, kidney slices were incubated for 15 min with increasing concentrations of L-DOPA (1-100 ILM) added to the medium. In experiments in which the effects of a-hANP (3.3 and 330 nM) and zaprinast (M&B 22,948; 10 PM), a relatively specific guanosine cyclic GMP phosphodiesterase inhibitor, were tested, the compounds were present during the preincubation and incubation periods. The preincubation and incubation were carried out in a shaking water bath at 370C, in an atmosphere of 95% 02 and 5% CO2; renal slices were incubated individually in glass vials containing 10 ml Krebs solution. After the incubation, renal slices were collected, washed for 30 min in ice cold Krebs solution, blotted with filter paper, minced with fine scissors and placed in 2 ml of 0.2 M perchloric acid at 40C for the next 24 h, before quantification of tissue catecholamines. Some experiments with kidney slices were performed with tissues taken from animals on a high salt diet (1% sodium chloride in the drinking fluid) during the previous 6 weeks. The daily sodium intake of rats on the high salt and normal salt diet averaged, respectively, 0.5 and 5 mmol 100 g-' body weight. In another series of experiments, kidney slices were obtained from 24-months old rats (considered to be old). In another set of experiments, kidney homogenates, instead of tissue slices, were used. Whole kidneys were homogenized in a modified Krebs solution with Duall-Kontes homogenizers and kept continuously on ice. The modified Krebs solution consisted of a medium similar to that described above with the exception that NaCl was reduced to 50 mM; the osmolarity of the medium was kept constant by the addition of 68 mM choline chloride. Aliquots of 1.0 ml of kidney homogenates plus 1.5 ml Krebs solution were placed in glass test tubes incubated for 60 min; thereafter, L-DOPA (0. 1 - 10.0 jM) was added to the medium for a further 15 min. In experiments in which the effect of x-hANP (330 nM) was tested, the peptide was present during the preincubation and incubation periods. During preincubation and incubation, kidney homogenates were continuously shaken and gassed (95% 02 and 5% CO2) at a constant temperature of 37TC. The reaction was stopped by adding 250 Al of 2 M perchloric acid and the preparations placed at 40C for 60 min; thereafter, kidney homogenates were centrifuged (2000 r.p.m., 2 min, 4°C) and aliquots of 1.5 ml of the supernatant used for the assay of L-DOPA, dopamine and DOPAC. The assay of L-DOPA, dopamine, noradrenaline and DOPAC in renal tissues and kidney homogenates was performed by means of high performance liquid chromatography with electrochemical detection, as previously described (Soares-da-Silva & Fernandes, 1991a). In brief, aliquots of 1.5 ml perchloric acid in which the tissues had been kept or 1.5 ml of supernatant of kidney homogenates were placed in 5 ml conical-based glass vials with 50 mg alumina and the pH of the samples immediately adjusted to pH 8.6 by the addition of Tris buffer. The adsorbed cate-
cholamines were then eluted from the alumina with 200 jl of 0.2 M perchloric acid on Millipore microfilters (MF 1); 50 gl of the eluate was injected into a high performance liquid chromatograph (Gilson Medical Electronics, Villiers le Bel, France). The lower limits for detection of L-DOPA, dopamine, noradrenaline and DOPAC were 1.0, 1.4, 0.9 and 2.5 pmol g- ', respectively. The protein content of the homogenates (mg of protein per g of tissue) was determined by the method of Lowry et al. (1951), with human serum albumin as a standard. Mean values ± s.e.mean of n experiments are given. Significance of differences between two means was estimated by Student's t test for unpaired data. Significance of difference between one control and several experimental groups was evaluated by the Tuckey-Kramer method (Sokal & Rohlf, 1981). A P value less than 0.05 was assumed to denote a significant difference. 3,4-Dihydroxyphenylacetic acid (DOPAC), L-dihydroxyphenylalanine (L-DOPA), dopamine hydrochloride, a-human atrial natriuretic peptide, (-)-a-methyl-p-tyrosine and noradrenaline bitartrate were purchased from Sigma Chemical Comany (St. Louis, Mo, U.S.A.) and zaprinast (M&B 22,948; 2-o-propoxyphenyl-8-azapurin-6-one) was kindly donated by the manufacturer (May & Backer, Ltd, Dagenham, Essex).
Results Incubation of renal tissues with 10 AM L-DOPA for 5, 10, 20 and 30 min or with increasing concentrations of L-DOPA for 15 min resulted, respectively, in a time- (Figure 1) and concentration-dependent (Figure 2) accumulation of newlyformed dopamine; the amount of the amine accumulated in the tissue reached values as high as 11 nmol g'- when 100 jAM L-DOPA was added to the medium (Figure 2). The accumulation of DOPAC, the deaminated metabolite of dopamine, in kidney slices was also dependent on the incubation time (Figure 1) and on the concentration of L-DOPA used (Figure 2) reaching values as high as 32 nmol g' when 100 AM LDOPA was used (Figure 2). In kidney slices incubated in the absence of exogenous L-DOPA, the dopamine and noradrenaline tissue concentrations were 0.018 ± 0.004 and 0.709 ± 0.041 nmol g-' (n = 8), respectively; DOPAC was not detectable under these experimental conditions. The tissue levels of noradrenaline in kidney slices did not significantly change in the course of these experiments even when 100 AM L-DOPA was used (data not shown, but see Fernandes & Soares-da-Silva, 1990). Incubation of kidney homogenates with increasing concentrations of L-DOPA (0.1-10.0 1M) for 15 min resulted in a concentrationdependent accumulation of newly-formed dopamine (Figure 3); the formation of DOPAC was dependent on the concentration of L-DOPA added to the medium (Figure 3). The addition of a-hANP (330 nM) to the incubation medium resulted in a marked decrease (63-78% reduction) in the time-dependent accumulation of dopamine and DOPAC in kidney slices incubated with 10 jM L-DOPA (Figure 1). The inhibitory effect of the peptide on the accumulation of newly-formed dopamine and DOPAC was also observed when 3.3 nM a-hANP was used; this only achieved statistical significance at 20 and 30 min of incubation (Figure 1). As shown in Figure 2, a-hANP (330 nM) was also found to decrease the accumulation of newly-formed dopamine in experiments in which increasing concentrations of L-DOPA were added to the medium during the 15 min incubation period. Under these experimental conditions, the inhibitory effect of a-hANP (45-66% reduction) could be observed at all concentrations of L-DOPA, even at 100 gM L-DOPA (Figure 2). The inhibitory effect of a-hANP on the accumulation of newly-formed dopamine was also accompanied by a reduction in the formation of DOPAC; this effect was similar (38-61% reduction) to that observed for
RENAL DOPAMINE SYNTHESIS AND ac-hANP
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L-DOPA (pM) Figure 2 Effect of a-human atrial natriuretic peptide (oc-hANP; 330 nM), zaprinast (10 pM) and x-hANP plus zaprinast on the accumulation of (a) dopamine and (b) 3,4-dihydroxyphenylacetic acid (DOPAC) (in nmol g-') in rat kidney slices incubated with increasing concentrations of exogenous L-DOPA (1-1I00 M) for 15 min. Each point represents the mean of five to six experiments per group; vertical lines show s.e.mean. Significantly different from corresponding values of control using the Tuckey Kramer method (*P
