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J Am Soc Nephrol 12:29-36, 2001
© 2001 American Society of Nephrology

Adenosine Activates Aromatic L-Amino Acid Decarboxylase Activity in the Kidney and Increases Dopamine

TAKANOBU TAKEZAKO, KEITA NODA, EMIKO TSUJI, MANABU KOGA, MANABU SASAGURI and KIKUO ARAKAWA

Department of Internal Medicine, Fukuoka University, School of Medicine, Fukuoka, Japan.

Correspondence to Dr. Keita Noda, Department of Internal Medicine, Fukuoka University, School of Medicine, 45-1-7 Nanakuma Jonan-ku Fukuoka City, Fukuoka, Japan, 814-0180. Phone: 81-92-801-1011 x3366; Fax: 81-92-865-2692; E-mail: keita{at}fukuoka-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. Renal sodium handling is important for regulating BP, and renal dopamine and adenosine play an important role in renal sodium handling, however the interaction of these hormones in the kidney was not clarified. In in vivo experiments, adenosine significantly increased water and sodium excretion by 50% compared with vehicle when infused into the left renal artery, accompanied by an increase in urinary dopamine excretion in the left kidney. Neither water-sodium excretion nor dopamine excretion changed in the vehicle-infused kidney. Aromatic L-amino acid decarboxylase activity in the left kidney was significantly higher than that in the noninfused right kidney. The increase in water-sodium excretion induced by adenosine was significantly inhibited by SCH23390, a selective D1 receptor antagonist. In in vitro experiments, porcine renal proximal tubular cells were incubated with 250 µM L-dopa and N6-cyclohexyladenosine, an adenosine type 1 receptor agonist, after treatment with adenosine deaminase. N6-cyclohexyladenosine significantly increased dopamine formation at a concentration of 10-9 to 10-7 M, and this was completely inhibited by 1,3-dipropyl-8-cyclopentylxanthin, an adenosine A1 antagonist. These results show that renal dopamine synthesis is stimulated by adenosine through the activation of aromatic L-amino acid decarboxylase and suggest that adenosine leads to an increase in renal dopamine and natriuresis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The renal sodium transport system plays an important role in regulating BP, and the impairment of renal sodium excretion causes hypertension. Among related factors, renal dopamine plays an important role in renal sodium excretion. Dopamine inhibits Na+-H+ exchange through the D1 receptor at the apical side and inhibits Na+-K+ ATPase at the basolateral side, resulting in the inhibition of sodium reabsorption (1). Although dopamine receptors are abundantly located in the proximal convoluted tubule (PCT), dopamine can also induce natriuresis in more distal nephron segments (1,2,3,4,5). PCT contains high levels of aromatic L-amino acid decarboxylase (AADC) (6,7), which converts L-dopa to dopamine (8). Other investigators and we reported that exercise increased urinary dopamine excretion accompanied by a reduction of BP (9,10,11). The mechanism of the increase in dopamine production by exercise is still unknown.

Adenosine is widely present in mammalian tissue and has various physiologic functions, including the regulation of cardiac rate, neurotransmitter release, smooth muscle tone, and platelet function (12). In the kidney, adenosine plays an important role in water-electrolyte metabolism, such as in the GFR, renal blood flow, renin release, tubuloglomerular feedback, tubular sodium and water transport, and neurotransmitter release (12). These effects seem to be mediated by adenosine type 1 (A1) and type 2 (A2) receptors. Recently, four subtypes, denoted A1, A2a, A2b, and A3, were found in renal cortex and medulla in the rat (13). However, subtype-specific effects on sodium handling in the kidney are controversial (14,15,16,17,18,19).

Adenosine is produced by two different enzymes, S-adenosylhomocysteine (SAH) hydrolase and 5'-nucleotidase. Intracellular adenosine is metabolized by three pathways: deamination to inosine by adenosine deaminase, phosphorylation to 5'-AMP by adenosine kinase, and coupling to SAH by SAH hydrolase (20). The local production of adenosine is reportedly increased with salt loading (21) and hypoxia (22,23,24,25,26). A recent study showed that the contraction of skeletal muscle increased the adenosine concentration in humans (23).

Both dopamine and adenosine are known to cause diuresis and natriuresis. It is possible that one of these might regulate the other in the kidney. On the basis of our previous observation that exercise increased dopamine excretion in the urine, we hypothesized that the adenosine increased by exercise up-regulates dopamine production in the kidney. In the present study, we attempted to clarify whether adenosine stimulates AADC activity through adenosine receptor and increases dopamine production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In Vivo Experiments
Animal Preparation. All animals received humane care in compliance with guidelines formulated by Fukuoka University. Male Sprague-Dawley rats (weight 360 to 500 g) that were fed normal rat chow and given free access to tap water were used for these studies. Rats were anesthetized by an intraperitoneal injection of 60 mg/kg body wt pentobarbital sodium (5-ethyl-5-[1-methylbutyl]-2,4,6-trioxo-hexahydro-pyrimidine sodium salt; Dainippon Pharmaceuticals, Osaka, Japan). Rats were placed on a thermostatically controlled heating table to maintain a rectal temperature of 37°C. Rats were tracheotomized and one 22-G Angiocath (Becton Dickinson, Franklin Lakes, NJ) was inserted into the right jugular vein for infusion, while another was inserted into the right femoral artery for continuous monitoring of BP and sampling of arterial blood. After an abdominal mid-line incision, the bilateral ureters were catheterized by a PE10 tube (Becton Dickinson) to collect urine separately from each kidney. A tapered PE10 tube was inserted into the left renal artery from the left carotid artery for the intrarenal infusion of drugs. After the catheter was inserted in the jugular vein, 5% bovine serum albumin (BSA) in saline was infused at a rate of 16.7 µl/100 g body wt per min during surgery to replace blood lost. After surgery, 5% (in protocol 1) or 2.5% (in protocol 2) BSA in saline was infused at a rate of 20 µl/100 g body wt per min to maintain a euvolic state. Inulin was added to the infusion solution at a final concentration of 5% to measure the GFR.

Protocol 1: Effect of Adenosine on Dopamine Synthesis in the Rat Kidney. After a 120-min equilibration period when constant urine flow rate (approximately 50 µl/min) was confirmed, urine was collected for 30 min during saline infusion at a rate of 10 µl/min into the left renal artery for basal measurement. After a 20-min equilibration period, adenosine (Sigma, St. Louis, MO) in saline was infused at a rate of 10 µg/min into the left renal artery, urine was collected for 30 min, and blood samples were taken at the end of each urine collection. After the experiment, bilateral kidneys were immediately removed and stored at -80°C until assayed for AADC activity.

Protocol 2: Effect of a Dopamine D1 Antagonist SCH23390 on the Natriuretic Effect of Adenosine in the Rat Kidney. After the basal measurement at the constant urine flow rate (30 to 50 µl/min) described above was taken, saline as a vehicle or adenosine (5 or 10 µg/min) was infused into the left renal artery. Finally, saline or adenosine (10 µg/min) was infused with 1 µg/min of SCH23390 (Funakoshi Co., Tokyo, Japan) into the left renal artery. A 20-min equilibration time was allowed between periods. During each period, urine was collected for 30 min. Blood samples were taken at the end of each urine collection.

Analytical Techniques. Urine volume was determined gravimetrically. Plasma and urine sodium concentrations were measured by flame photometry (model-710, Hitachi, Tokyo, Japan). Inulin concentrations in plasma and urine were determined by the anthrone method (27). The dopamine concentration in urine was measured by HPLC (28).

AADC activity in the renal cortex was determined by the rate of conversion of L-dopa (Sigma) to dopamine. AADC activity was determined as described previously (29). The renal cortex was collected and homogenized (1:9, wt/vol) in solution consisting of 0.25 M sucrose, 0.1 mM toluenesulfonyl fluoride, 1 mM ethylenediamine tetraacetic acid disodium (EDTA-Na2), and 10 mM Tris-HCl (pH 7.5). The homogenate was then centrifuged at 16,000 x g for 60 min. The incubation medium consisting of 30 mM NaH2PO4, 30 mM Na2HPO4, 0.3 mM EDTA-Na2, 0.17 mM ascorbic acid, 0.01 mM pyridoxal phosphate, 0.1 mM pargyline hydrochloride, and 0.1 mM toluenesulfonyl fluoride was prewarmed at 37°C for 5 min, and 10 µl of sample and 25 µl of 8 mM L-dopa were added. After incubation at 37°C for 30 min, 400 µl of 0.2 N perchloric acid (PCA) was added to stop the enzymatic reaction. The dopamine concentration in the reaction mixture was measured by HPLC. Protein contents in tissue homogenates were determined by the BCA method (30). AADC activity was expressed as picomoles of dopamine formed per milligram of protein per minute. GFR was calculated as the product of urine flow and the urine-to-plasma inulin concentration ratio.

In Vitro Experiments
Cell Culture and Preparation. The LLC-PK1 cell line, a porcine-derived renal proximal tubular cell line (American Type Culture Collection, Rockville, MD), was grown in Medium 199 (Life Technologies, Rockville, MD) supplemented with 5% fetal bovine serum (ICN, Costa Mesa, CA) in humidified 95% air and 5% CO2 at 37°C. For these studies, cells were seeded in 6-well plastic culture clusters (diameter 35 mm) (Costar, Corning, NY) and cultured for 2 d after confluence. Each experiment was performed 24 h after cells were incubated in serum-free medium.

Before each experiment, the cells were washed twice with 2 ml of Krebs-Ringer bicarbonate medium (KRB) (31), which consisted of 118 mM NaCl, 4.85 mM KCl, 2.5 mM CaCl2, 1.15 mM MgSO4, 1.15 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM glucose (pH 7.4). The cells were incubated at 37°C for 30 min in 2 ml of KRB containing L-dopa with or without other experimental reagents. After the incubation period, the KRB was rapidly removed and 0.2 N PCA was added to the culture plate. Dopamine in KRB reflected dopamine release from the cells, and dopamine in PCA extracts was considered intracellular dopamine. The dopamine concentrations in KRB and PCA extract were determined by HPLC (28). Total dopamine synthesis was determined as the sum of the amount of extra- and intracellular dopamine.

Ability of LLC-PK1 Cells to Synthesize and Release Dopamine. We performed two pilot studies: a dose-response study and a time-course study. Cells were incubated with various concentrations of L-dopa (0 to 500 µM) for 30 min and incubated with L-dopa (250 µM) for various times to determine the appropriate conditions for the following experiments.

Influence of Adenosine Metabolism in LLC-PK1 Cells. Because of the short half-life (T1/2 = 1 to 2 s) of adenosine in vitro metabolism by cells, we examined the time course of the metabolism of exogenous adenosine (10-6 M) by cells incubated in adenosine-free KRB for 30 min. The adenosine concentration was determined by RIA (32). To evaluate the effect of endogenous adenosine released from LLC-PK1 cells on dopamine synthesis, we incubated cells for 30 min with 250 µM L-dopa in the presence or absence of adenosine deaminase (ADA; 0.5 U/ml; Sigma) (33).

Effects of Adenosine Receptor Agonist and Antagonist on Dopamine Synthesis in LLC-PK1 Cells. To determine the adenosine receptor subtype that is related to dopamine synthesis, we incubated cells for 30 min with N6-cyclohexyladenosine (CHA; Sigma) (10-11 to 10-5 M), a selective adenosine receptor type 1 (A1) agonist in the presence or absence of 1,3-dipropyl-8-cyclopentylxanthin (DPCPX; Sigma) (10-6 M), a selective A1 antagonist. Before the dose-response experiment, cells were pretreated with 0.5 U/ml ADA to destroy the endogenous adenosine released from cells.

Western Blot Analysis. The membrane fraction of LLC-PK1 cells was prepared by freeze and thaw methods. Twenty-five µg of membrane fractions were diluted with one-half volume of sample buffer that contained 4% sodium dodecyl sulfate (SDS), 20% glycerol, and 2% 2-mercaptoethanol in 125 mM Tris-HCl buffer (pH 6.8). Polyacrylamide gel electrophoresis (PAGE) was performed in the presence of SDS. After SDS-PAGE, electrophoretic transfer was performed using a transfer membrane (Millipore Co., Bedford, MA). The blotted membrane was blocked in TBS (50 mM Tris, 150 mM NaCl [pH 7.4]) containing 0.5% blocking reagent and 0.05% Tween 20. The membrane was incubated with preimmune serum or anti-A1 receptor antiserum (Oncogene Research Products, Cambridge, MA; 1:1,000) and then incubated with anti-rabbit Ig antibody conjugated horseradish peroxidase (Bio-Rad, Hercules, CA; 1:2,000). The detection reagents of ECLTM (Amersham Pharmacia Biotech, Buckinghamshire, UK) was added, and the membrane was incubated for 1 min. The membrane was exposed to x-ray film (Amersham Pharmacia Biotech) for 20 s.

Statistical Analyses
Data are expressed as the mean ± SEM. The significance of differences between the groups was assessed by a one-way ANOVA for repeated measures, and Fisher's protected least significant difference procedure was used to detect which values were different from the control values. Simple comparisons between the two groups were performed using the paired and unpaired t test. P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In Vivo Experiments
Effect of Adenosine on Dopamine Synthesis in the Rat Kidney. The infusion of adenosine into the left renal artery significantly increased urine flow by 52.5 ± 11.8%, sodium excretion by 28.3 ± 11.4%, and dopamine excretion by 24.1 ± 6.6% compared with saline infusion; AD 0 (Table 1). Urine flow, sodium excretion, and dopamine excretion in the right kidney (noninfused side) were not affected when adenosine was infused into the left renal artery. AADC activity in the renal cortex was significantly higher in the left kidney, by 47.5 ± 16.9%, compared with that in the right kidney (Figure 1).


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  		Table 1. Effect of intrarenal infusion of adenosine on renal functiona
 


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  		Figure 1. Effect of intrarenal infusion of adenosine on aromatic L-amino acid decarboxylase (AADC) activity in the renal cortex. AADC activity was measured after adenosine infusion at 10 µg/min for 50 min into the left renal artery. {square}, right kidney;, left kidney (adenosine-infused side). Data are mean ± SEM (n = 6); *, P < 0.05 versus right kidney, by unpaired t test.

 

Effect of a Dopamine D1 Receptor Antagonist on the Natriuretic Effect of Adenosine in the Rat Kidney. Because adenosine caused diuretic and natriuretic effects with an increase in dopamine excretion in protocol 1, we next investigated whether these diuretic and natriuretic effects were caused by the increase of renal dopamine. The infusion of adenosine into the left renal artery dose-dependently increased urine flow, sodium excretion, and dopamine excretion, whereas vehicle infusion did not compared with saline infusion (basal period; Figure 2 and Table 2). A dopamine D1 receptor antagonist, SCH23390, significantly decreased urine flow by 64% and completely inhibited sodium excretion compared with adenosine infusion (10 µg/min). However, SCH23390 alone did not influence urine volume, sodium excretion, and dopamine secretion.



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  		Figure 2. Effect of intrarenal infusion of saline (vehicle), adenosine, or SCH23390 (SCH) on urine flow (A), sodium (B), and dopamine excretion (C). AD 0 represents infusion of saline. AD 5, AD 10, SCH, and AD 10+SCH represent 5 µg/min of adenosine infusion, 10 µg/min of adenosine infusion, 1 µg/min of SCH23390, and 10 µg/min of adenosine infusion with 1 µg/min of SCH23390, respectively, into the left renal artery. {square}, vehicle infusion (n = 6); {blacksquare}, adenosine infusion (n = 7). Data are mean ± SEM; *, P < 0.05 versus AD 0 or saline (basal period); **, P < 0.05 versus AD10.

 

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  		Table 2. Effect of intrarenal infusion of adenosine and adenosine plus SCH23390 on renal functiona
 

GFR in the left kidney was not significantly changed during the infusion of vehicle, adenosine, or adenosine plus SCH23390 (Table 2). MAP was not changed during each period (data not shown).

In Vitro Experiments
Changes in Dopamine Synthesis in LLC-PK1 Cells with Various Concentrations of L-dopa and with Various Incubation Times. Incubation of LLC-PK1 cells with various concentrations of L-dopa for 30 min produced dose-dependent increases in intracellular, extracellular, and total dopamine synthesis (Figure 3). In the time-course study, the intracellular, extracellular, and total synthesis of dopamine all increased with time (Figure 4). These results suggest that the LLC-PK1 cell line was a useful model for studying renal dopamine synthesis. Therefore, we used this cell line for the following experiments with 250 µM L-dopa as a substrate concentration in the medium and 30 min as an incubation time to determine dopamine synthesis.



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  		Figure 3. Dose-dependent effect of L-dopa on the intracellular ({blacktriangleup}), extracellular ({blacksquare}), and total synthesis ([UNK]) of dopamine in LLC-PK1 cells (n = 2). Cells were incubated with various concentrations of L-dopa for 30 min in a 35-mm well. Data are mean ± SEM.

 


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  		Figure 4. Time-dependent effect of L-dopa on the intracellular ({blacktriangleup}), extracellular ({blacksquare}), and total synthesis ([UNK]) of dopamine in LLC-PK1 cells. The cells were incubated in the presence of 250 µM L-dopa for various incubation times (n = 2). Data are mean ± SEM.

 

Influence of Adenosine Metabolism in LLC-PK1 Cells. Exogenously added adenosine in fresh KRB (final concentration, 1000 nM) decreased rapidly and markedly to 41.4 ± 11.2 nM in the medium after only 5 s of incubation. However, after 30 min of incubation, the extracellular adenosine concentration had increased to 659.8 ± 35.8 nM. This result suggests that exogenous adenosine was rapidly taken up into the starved cells under the condition of free adenosine in the medium, and then adenosine was released from cells into the medium.

Pretreatment with ADA, which destroys adenosine, attenuated dopamine synthesis by 63.6 ± 2.1% compared with that without ADA. These results suggest that endogenous adenosine stimulates the synthesis and release of dopamine in LLC-PK1 cells. Therefore, we decided to minimize the effect of endogenous adenosine by adding ADA before the experiments and to use a synthetic analogue as an adenosine receptor agonist.

Effect of Adenosine Receptor Agonist and Antagonist on Dopamine Synthesis in LLC-PK1 Cells. CHA, an A1 receptor agonist, at a concentration of 10-9 M increased the extracellular, intracellular, and total synthesis of dopamine by 39.3 ± 7.1, 11.0 ± 3.7, and 23.0 ± 5.2%, respectively, compared with treatment with the vehicle alone. These increases were completely abolished by 10-6 M of DPCPX, an A1 receptor antagonist (Figure 5). However, a concentration of CHA above 10-6 M had no effect on dopamine synthesis. These results suggest that the stimulatory effect of adenosine on dopamine synthesis is mediated by adenosine A1 receptor.



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  		Figure 5. Stimulatory effect of N6-cyclohexyladenosine (CHA) on dopamine synthesis and the inhibitory effect of 1,3-dipropyl-8-cyclopentylxanthin (DPCPX) on that effect of CHA in LLC-PK1 cells (n = 6, respectively). The cells were preincubated with 0.5 U/ml adenosine deaminase (ADA) for 30 min. After adding 250 µM L-dopa, the cells were incubated with various concentrations of CHA in the absence ([UNK]) or presence ({blacktriangleup}) of DPCPX for 30 min. Data are mean ± SEM. *, P < 0.05 versus in the absence of CHA ({circ}); **, P < 0.05 versus in the absence of CHA and presence of 10-6 M DPCPX ({triangleup}).

 

Western Blot Analysis. Approximately 25 µg of membrane fractions of LLC-PK1 cells was applied to lanes 1 and 2. One major band at a molecular mass of 38 kD was evident on lane 2 (Figure 6).



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  		Figure 6. Western blot analysis of the membrane fraction of LLC-PK1 cell homogenates. Lanes 1 and 2 were immunoblotted by preimmune serum and anti-A1 receptor antibody, respectively. Arrow shows the presence of A1 receptor compatible with a molecular mass of 38 kD.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report that the administration of adenosine increased dopamine synthesis accompanied by an increase in AADC activity in the kidney as well as in LLC-PK1 cells. AADC is a key enzyme in the conversion of L-dopa to the biologically active chemical mediator dopamine, and our results showed an increase in urinary dopamine secretion in association with the activation of AADC. The present study showed that the infusion of adenosine to the renal artery increased urine flow and sodium excretion in the infused kidney. The natriuretic effect without any change in GFR suggests that adenosine does not have an effect through hydrodynamics but rather has a direct action on renal tubular cells.

Natriuretic or antinatriuretic effects of adenosine were previously reported in in vivo experiments. The administration of an A1 antagonist into the vein or interstitium of the renal medulla induced natriuresis (16,34). However, Yagil (14) showed that adenosine infusion via a renal artery induced natriuresis, whereas adenosine infusion via the aorta did not change sodium excretion. In addition, Fransen and Koomans (15) showed that natriuresis was induced by the administration of adenosine via the renal artery, whereas the aortic infusion of adenosine produced antinatriuresis mediated by stimulating renal nerves. These results are compatible with those of our present study. This discrepancy in the effects of adenosine on sodium handling might depend on the systemic effects of adenosine on hemodynamics and the neural system.

It is surprising that the present study showed that increased sodium excretion induced by adenosine was completely blocked by a dopamine D1 receptor antagonist, SCH23390. Increased urine flow induced by adenosine was blocked by 64% by blocking D1 receptor. These results suggested that the increase in water excretion was mainly, and the increase in sodium excretion was totally, dependent on dopamine synthesis stimulated by the administration of adenosine into the kidney rather than a direct effect of adenosine on sodium channels of tubular cells. Approximately half of the increased urine flow, which is not inhibited by D1 receptor antagonist, may be caused by a direct effect of adenosine (34).

Next, we performed an in vitro experiment using the LLC-PK1 cell line, a porcine-derived proximal tubule cells, which is reported to express both AADC (31,35) and adenosine receptors (36). These cells have the ability to synthesize adenosine, release it extracellularly, and take it up into cells, finally reaching an equilibrium state in the culture condition. This results in the production of sufficient adenosine to stimulate adenosine receptor signaling. This might be the reason that many studies (36,37,38,39,40) have used an extremely high concentration of adenosine as an A1 agonist in cell culture experiments. Although we also used a high concentration of adenosine to stimulate the receptor on LLC-PK1 cells, no increase in dopamine was observed (data not shown). However, in the presence of ADA to inactivate endogenous adenosine, dopamine synthesis was increased by CHA, a synthetic A1 agonist at a concentration near its Kd (0.3 to 3 nM) for A1 receptor in LLC-PK1 cells. CHA is a metabolically stable adenosine agonist (41) that is not taken up into cells. In a dose-response study, CHA increased the extracellular release and total synthesis of dopamine at a low concentration (10-9 to 10-7M), which was enough to occupy almost all of the A1 receptors expressed on the cell surface. Furthermore, these effects were completely inhibited by 10-6 M DPCPX, an A1 adenosine receptor antagonist. The expression of A1 receptor in this cell line was confirmed by Western blot in the present study.

Conversely, CHA did not seem to have any stimulatory effects on the release and synthesis of dopamine at a high concentration (10-6 to 10-5 M; Figure 5). The disappearance of a stimulatory effect of CHA at a high concentration could be due to an A2 receptor-mediated phenomenon, because 10-6 to 10-5 M of CHA can bind and stimulate adenosine A2a receptor (Kd value of CHA for adenosine A2a receptor is 200 to 500 nM). These results suggest that the stimulatory effect of the release and synthesis of dopamine was caused by the activation of adenosine A1 receptor under these physiologic conditions.

Signal transduction pathways of adenosine A1 receptor are known to inhibit adenylate cyclase, activate phospholipase C, and increase inositol triphosphate and cytosolic Ca2+ (33,42). Conversely, AADC activity is regulated by a coenzyme (pyridoxine), intracellular pH (43), and Ca2+ concentration (44); however, it is not clear how the intracellular signaling of adenosine receptor regulates AADC activity.

With regard to the origin of adenosine in the kidney, it is thought to be derived from two pathways. In the first pathway, adenosine is filtered from the systemic circulation. A recent report (23) showed that adenosine increased approximately fivefold and its precursors, ATP, ADP, and AMP, markedly increased 15- to 30-fold in the interstitium of contracting skeletal muscle during exercise in humans; adenosine is believed to originate from nerve endings. Regarding filtration into the tubular lumen, when radiolabeled adenosine was injected into the renal artery, 53% of the adenosine was filtered and recovered in the urine and 11% of adenosine was recovered in the venous plasma (45). Taken together, these results suggest that increased plasma adenosine and its precursors may be filtered into tubules. As a second pathway, adenosine is locally produced in the tubules. There is ample evidence that cellular nucleotides locally released from nerve endings are converted to adenosine by ecto-5'-nucleotidase present at extracellular sites along the nephron including the cortical collecting tubules (46). It has been shown that an endogenous cAMP-adenosine pathway is present in the rat kidney. Stimulation of the renal sympathetic nerve and ß adrenoceptor could increase the secretion of adenosine from cAMP by stimulating 5'-nucleotidase (47,48).

In conclusion, the intrarenal infusion of adenosine induced diuresis and natriuresis in association with an increase in AADC activity and activation of the renal dopamine system in in vivo rat kidney. Adenosine is endogenously produced in porcine-derived proximal tubular cell, which also increases dopamine synthesis by activating AADC through adenosine A1 receptor activation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication November 30, 1999. Accepted for publication May 16, 2000.




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