Adenosine A_2A receptor activation attenuates tubuloglomerular feedback responses by stimulation of endothelial nitric oxide synthase

Abstract

Adenosine A₂ receptors have been suggested to modulate tubuloglomerular feedback (TGF) responses by counteracting adenosine A₁ receptor-mediated vasoconstriction, but the mechanisms are unclear. We tested the hypothesis that A_2A receptor activation blunts TGF by release of nitric oxide in the juxtaglomerular apparatus (JGA). Maximal TGF responses were measured in male Sprague-Dawley rats as changes in proximal stop-flow pressure (ΔP_SF) in response to increased perfusion of the loop of Henle (0 to 40 nl/min) with artificial tubular fluid (ATF). The maximal TGF response was studied after 5 min intratubular perfusion (10 nl/min) with ATF or ATF + A_2A receptor agonist (CGS-21680; 10⁻⁷ mol/l). The interaction with nitric oxide synthase (NOS) isoforms was tested by perfusion with a nonselective NOS inhibitor [N^ω-nitro-l-arginine methyl ester hydrochloride (l-NAME); 10⁻³ mol/l] or a selective neuronal NOS (nNOS) inhibitor [N^ω-propyl-l-arginine (l-NPA); 10⁻⁶ mol/l] alone, and with the A_2A agonist. Blood pressure, urine flow, and P_SF at 0 nl/min were similar among the groups. The maximal TGF response (ΔP_SF) with ATF alone (12.3 ± 0.6 mmHg) was attenuated by selective A_2A stimulation (9.5 ± 0.4 mmHg). l-NAME enhanced maximal TGF responses (18.9 ± 0.4 mmHg) significantly more than l-NPA (15.2 ± 0.7 mmHg). Stimulation of A_2A receptors did not influence maximal TGF response during nonselective NOS inhibition (19.0 ± 0.4) but attenuated responses during nNOS inhibition (10.3 ± 0.4 mmHg). In conclusion, adenosine A_2A receptor activation attenuated TGF responses by stimulation of endothelial NOS (eNOS), presumably in the afferent arteriole. Moreover, NO derived from both eNOS and nNOS in the JGA may blunt TGF responses.

the tubuloglomerular feedback (TGF) mechanism is a negative feedback loop that enhances vascular tone of the afferent arteriole when tubular NaCl delivery at the macula densa is increased (44); thus, TGF contributes to renal autoregulation and blood pressure control. Osswald and coworkers (30) first proposed that local generation of adenosine, as a consequence of increased NaCl transport, may elicit TGF-induced afferent arteriole vasoconstriction (30). Adenosine is a paracrine agent that signals through G protein-coupled A₁, A₂, and A₃ receptors. The adenosine A₂ receptor family consists of two subtypes, A_2A and the A_2B, that possess a high and a low agonist affinity, respectively (51). Adenylate cyclase, thus cAMP production, is stimulated by the adenosine A_2A and A_2B receptors but inhibited by A₁ and A₃ receptors (51). Studies in gene-deficient mice, or with receptor antagonists, have reported that TGF is mediated via activation of adenosine A₁ receptors (3, 8, 45, 46).

Adenosine receptors are expressed in renal afferent arterioles (17, 22, 51, 53) and regulate preglomerular resistance via A₁ receptor-mediated constriction and A₂ receptor-mediated dilatation (12, 22). In a recent study, we demonstrated that adenosine A₂ receptors modulate the TGF response by counteracting adenosine A₁ receptor-mediated vasoconstriction (6). However, the mechanisms for A₂ receptor-mediated dilatation of afferent arterioles and its modulation of TGF are not established. Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS or NOS3) modulates the contractile response of afferent arterioles (15, 21, 31, 40, 50), whereas neuronal nitric oxide synthase (nNOS or NOS1) in the macula densa is considered the primary NOS isoform modulating the TGF response (39, 48, 52, 55, 57). Inducible nitric oxide synthase (iNOS or NOS2) is not considered important in regulation of arteriolar contraction or TGF responses in normal rats (32, 48). In the present study, we tested the hypothesis that activation of adenosine A_2A receptors blunts the TGF response by increasing NO production from eNOS and/or nNOS in the juxtaglomerular apparatus (JGA).

MATERIALS AND METHODS

Animals

This study was approved by the Georgetown University Animal Care and Use Committee and performed according to the National Institutes of Health guidelines for the conduct of experiments in animals. Male Sprague-Dawley rats (Charles River Laboratories International) weighing 350–450 g were maintained on a standard rat chow (0.3 g sodium/100 g content) with free access to food and tap water until the day of the study.

Surgical Preparation

On the day of the experiment, rats were anesthetized by an intraperitoneal injection of thiobarbital (Inactin, 120 mg/kg body wt; Research Biochemicals, Natick, MA) and were placed on a servo-regulated heating pad to maintain body temperature at 37°C. The trachea was cannulated to allow spontaneous breathing. Catheters were placed in the left jugular vein for infusion of maintenance fluid (0.9% NaCl; 5 ml·h⁻¹·kg body wt⁻¹) and in the right femoral artery for the recording of mean arterial pressure (MAP). The bladder and the left ureter were cannulated to ensure unrestricted urine flow. The left kidney was exposed by a flank incision, cleaned from surrounding tissue stabilized in a Lucite cup without stretching the renal vessels, and bathed in 0.9% NaCl solution maintained at 37°C. Micropuncture experiments were initiated after a 30- to 40-min stabilization period.

TGF Experiments

TGF was determined by the stop-flow technique. Under a stereomicroscope, randomly chosen proximal tubular segments on the kidney surface were punctured with a sharpened micropipette (7–9 μm OD) containing artificial tubular fluid [ATF; (in mM) 128 NaCl, 4 NaHCO₃, 5 KCl, 2 CaCl₂, 7 urea, and 2 MgCl₂, pH 7.4] stained with Lissamine green dye (2 g/l) to identify the nephron and the direction of the tubular flow. Subsequently, grease (T grade; Apiezon, Manchester, UK) was inserted in the micropuncture site with a micropipette (7–9 μm OD) connected to a hydraulic drive (Trent Wells, La Jolla, CA) to halt tubular flow. A perfusion pipette containing ATF with testing compounds or vehicle was inserted in the late proximal tubule downstream from the grease block and connected to a calibrated nanoliter microperfusion pump (Vestavia Scientific, Birmingham, AL). A pressure pipette (3–5 μm OD) was inserted in the proximal tubule upstream from the grease block to measure proximal stop-flow pressure (P_SF) or in unobstructed nephrons to determine proximal free-flow pressure (P_FF). The micropressure system (model 900A; World Precision Instruments, Sarasota, FL) was connected to a Powerlab (AD Instruments, Colorado Springs, CO) to record MAP, P_FF, and P_SF.

Study Design

The maximal TGF response (ΔP_SF) for each nephron was assessed by the difference in P_SF at zero loop perfusion and during perfusion at 40 nl/min for 2 min. The adenosine receptor agonist was dissolved in DMSO and added to ATF. The final concentration of DMSO was 1%, which in preliminary experiments did not affect TGF. The stop-flow measurements were taken before and after 5 min intratubular administration at a low, non-TGF-activating perfusion rate (i.e., 10 nl/min). The protocols were as follows: protocol 1, A_2A receptor agonist [4-(2-{[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino}ethyl)benzenepropanoic acid hydrochloride (CGS-21680); 10⁻⁷ mol/l]; protocol 2, nonselective nitric oxide synthase (NOS) inhibitor [N^ω-nitro-l-arginine methyl ester hydrochloride (l-NAME); 10⁻³ mol/l]; protocol 3, CGS-21680 (10⁻⁷ mol/l) + l-NAME (10⁻³ mol/l); protocol 4, selective nNOS inhibitor [N^ω-propyl-l-arginine (l-NPA); 10⁻⁶ mol/l]; and protocol 5, CGS-21680 (10⁻⁷ mol/l) + l-NPA (10⁻⁶ mol/l).

Pretreatment and Drugs

In the experimental approach used in this and similar studies, the concentrations of the perfused compounds (adenosine analogs or NOS inhibitors) at the effector site are unknown but are likely below the perfusate concentrations. To avoid substantial dilution and to allow time for transportation to the effector site, intratubular perfusion was performed before the maximal TGF response was assessed.

CGS-21680.

CGS-21680, purchased from Tocris Bioscience (Ellisville, MO) (catalog no. 1063), is a potent and highly selective adenosine A_2A agonist and is active in vivo. It has binding affinities of 290, 27, 88,800, and 67 nM for A₁, A_2A, A_2B, and A₃ receptors, respectively (20, 27, 28, 34). CGS-21680 therefore exhibits ∼10-fold, 3,300-fold, and 2.5-fold selectivity for the A_2A receptor vs. A₁, A_2B, and A₃ receptors, respectively. The dose of CGS-21680 used in the present study (i.e., 10⁻⁷ mol/l) should predominantly inhibit A_2A but may also influence A₃ receptors. However, A₁ and A_2B receptors should not be affected.

l-NAME.

l-NAME, purchased from Sigma-Aldrich (Sigma Chemical, St. Louis, MO) (catalog no. N5751) is hydrolyzed in vivo to form the functional inhibitor N^ω-nitro-l-arginine (l-NNA). It has binding affinities of 15, 39, and 4,400 nM for nNOS, eNOS, and iNOS, respectively (10, 11, 25). l-NNA therefore exhibits similar selectivity for nNOS and eNOS but is ∼220-fold more selective for the constitutive vs. inducible NOS. The dose of l-NAME used in the present study (i.e., 10⁻³ mol/l) should inhibit all NOS isoforms.

l-NPA.

l-NPA (10⁻⁶ mol/l), purchased from Cayman Chemical (Ann Arbor, MI) (catalog no. 80587), is a potent and selective inhibitor of nNOS and is active in vivo. It has binding affinities of 57, 8,500, and 180,000 nM for nNOS, eNOS, and iNOS, respectively (2, 18, 58). l-NPA therefore exhibits ∼3,150-fold and 150-fold selectivity for the neuronal isoform vs. the inducible and endothelial isoforms of NOS, respectively. The dose of l-NPA used in the present study (i.e., 10⁻⁶ mol/l) should only inhibit nNOS.

Statistics

Values are presented as means ± SE. Single comparisons between normally distributed parameters were tested for significance with Student's paired or unpaired t-test. For the stop-flow pressure measurements, multiple groups were compared by one-way ANOVA. The Bonferroni post test for paired multiple comparisons was used to allow for more than one comparison with the same variable. This states a significance level of P/M, where M is the number of comparisons to be made. Statistical significance was defined as P < 0.05.

RESULTS

All animals were in good condition, and, at the time of experiments, there were no differences in body weights (370 ± 20 g) among the groups. Blood pressure, urine flow (12.4 ± 0.7 nl·min⁻¹·g body wt⁻¹), P_FF, and P_SF at 0 nl/min were similar among the groups (Table 1).

Table 1.

Blood pressure and proximal free-flow and stop-flow pressures

	Control	CGS-21680	L-NAME	CGS-21680 + L-NAME	L-NPA	CGS-21680 + L-NPA
MAP, mmHg	104 ± 3	106 ± 4	108 ± 5	99 ± 7	96 ± 6	100 ± 4
PFF, mmHg	10.4 ± 0.4	10.4 ± 0.5	10.5 ± 0.6	10.0 ± 0.6	10.5 ± 0.5	10.0 ± 0.8
PSF, mmHg	38.7 ± 1.0	36.1 ± 0.9	39.3 ± 1.9	37.8 ± 1.9	39.2 ± 1.7	38.4 ± 1.3
m/n	6/12	4/10	3/9	3/9	4/6	4/10

Values are means ± SE. Control, artificial tubular fluid; CGS-21680, A_2A receptor agonist 4-(2-{[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino}ethyl)benzenepropanoic acid hydrochloride (10⁻⁷ mol/l); L-NAME, nonselective NOS inhibitor N^ω-nitro-l-arginine methyl ester hydrochloride (10⁻³ mol/l); L-NPA, selective nNOS inhibitor N^ω-propyl-l-arginine (10⁻⁶ mol/l);

MAP, mean arterial pressure; P_FF, proximal free-flow pressure; P_SF, proximal stop-flow pressure at 0 nl/min perfusion; m/n, no. of animals and nephrons, respectively.

Effect of Adenosine A_2A Receptor Agonist (CGS-21680)

The control TGF response (ΔP_SF) was 12.3 ± 0.6 mmHg (Fig. 1A). After intratubular perfusion with A_2A receptor agonist (10⁻⁷ mol/l), the maximal TGF response was clearly attenuated (9.5 ± 0.4 mmHg) compared with control (Fig. 1B). Examples of original recordings of P_SF are shown in Fig. 1.

Fig. 1.

Top: change in proximal stop-flow pressure (ΔP_SF) in response to increased perfusion of loop of Henle from 0 to 40 nl/min. Broken lines represent individual nephrons. The maximal tubuloglomerular feedback (TGF) response was studied after 5 min intratubular perfusion (10 nl/min) with artificial tubular fluid (ATF) alone (control; m/n = 6/12) (A) or in combination with A_2A receptor agonist [4-(2-{[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino}ethyl)benzenepropanoic acid hydrochloride (CGS-21680); m/n = 4/10] at 10⁻⁷ mol/l (B). m/n, No. of animals and nephrons, respectively. *P < 0.05 compared with proximal stop-flow pressure (P_SF), at 0 nl/min, within each group (paired t-test). Bottom: sample recordings of mean arterial pressure (MAP) and P_SF during loop of Henle perfusion with ATF alone (control) (A) or with A_2A receptor agonist (CGS-21680) at 10⁻⁷ mol/l (B). Gray boxes represent a loop of Henle perfusion flow of 40 nl/min for 2 min.

Effect of Adenosine A_2A Receptor Agonist (CGS-21680) During Nonselective NOS Inhibition

Intratubular perfusion with l-NAME (10⁻³ mol/l) significantly enhanced the maximal TGF response (18.9 ± 0.4 mmHg) (Fig. 2A) compared with control (12.3 ± 0.6 mmHg; see Fig. 1A). Perfusion with CGS-21680 to stimulate A_2A receptors during simultaneous NOS inhibition did not attenuate the maximal TGF response (19.0 ± 0.4 mmHg) (Fig. 2B) compared with l-NAME alone (18.9 ± 0.4 mmHg). Examples of original recordings of P_SF are shown in Fig. 2.

Fig. 2.

Top: ΔP_SF in response to increased perfusion of loop of Henle from 0 to 40 nl/min. Broken lines represent individual nephrons. The maximal TGF response was studied after 5 min intratubular perfusion (10 nl/min) with the nonselective nitric oxide synthase (NOS) inhibitor [N^ω-nitro-l-arginine methyl ester hydrochloride (l-NAME); m/n = 3/9] at 10⁻³ mol/l alone (A), or in combination with A_2A receptor agonist (CGS-21680; m/n = 3/9) at 10⁻⁷ mol/l (B). m/n, No. of animals and nephrons, respectively. *P < 0.05 compared with P_SF, at 0 nl/min, within each group (paired t-test). Bottom: sample recordings of MAP and P_SF during loop of Henle perfusion with nonselective NOS inhibitor (l-NAME) at 10⁻³ mol/l alone (A) or in combination with A_2A receptor agonist (CGS-21680) at 10⁻⁷ mol/l (B). Gray boxes represent a loop of Henle perfusion flow of 40 nl/min for 2 min.

Effect of Adenosine A_2A Receptor Agonist (CGS-21680) During Selective nNOS Inhibition

Intratubular perfusion with l-NPA (10⁻⁶ mol/l) to inhibit nNOS specifically enhanced the maximal TGF response (15.2 ± 0.7 mmHg) (Fig. 3A) compared with control (12.3 ± 0.6 mmHg; see Fig. 1A). Perfusion with CGS-21680 to stimulate A_2A receptors during simultaneous nNOS inhibition attenuated the maximal TGF response (10.3 ± 0.4 mmHg) (Fig. 3B) compared with l-NPA alone (15.2 ± 0.7 mmHg). Examples of original recordings of P_SF are shown in Fig. 3.

Fig. 3.

Top: ΔP_SF in response to increased perfusion of loop of Henle from 0 to 40 nl/min. Broken lines represent individual nephrons. The maximal TGF response was studied after 5 min intratubular perfusion (10 nl/min) with selective nNOS inhibitor [N^ω-propyl-l-arginine (l-NPA); m/n = 4/6] at 10⁻⁶ mol/l alone (A) or in combination with A_2A receptor agonist (CGS-21680; m/n = 4/10) at 10⁻⁷ mol/l (B). m/n, No. of animals and nephrons, respectively. *P < 0.05 compared with P_SF, at 0 nl/min, within each group (paired t-test). Bottom: sample recordings of MAP and P_SF during loop of Henle perfusion with selective nNOS inhibitor (l-NPA) at 10⁻⁶ mol/l alone (A) or in combination with A_2A receptor agonist (CGS-21680) at 10⁻⁷ mol/l (B). Gray boxes represent a loop of Henle perfusion flow of 40 nl/min for 2 min.

A summary of the maximal TGF responses for the different experimental protocols is shown in Fig. 4. Stimulation of A_2A receptors (CGS-21680) attenuated TGF responses compared with that of ATF alone (control). Both nonselective NOS inhibition (l-NAME) and selective nNOS inhibition (l-NPA) increased the TGF response. However, the degree of TGF enhancement was less with the nNOS inhibitor. The A_2A agonist-mediated attenuation of TGF was abolished by nonselective NOS inhibition but was unaffected by nNOS inhibition.

Fig. 4.

Summary of maximal TGF responses (ΔP_SF) during ATF perfusion (control) (A), nonselective NOS inhibition (l-NAME; 10⁻³ mol/l) (B), selective nNOS inhibition (l-NPA; 10⁻⁶ mol/l) (C) alone (black bars), or together with adenosine A_2A receptor agonist (CGS-21680; 10⁻⁷ mol/l) (gray bars). Values are means ± SE. *P < 0.05 compared with control. #P < 0.05 compared with l-NPA alone.

DISCUSSION

In the present study, using the stop-flow technique to determine TGF, we show that stimulation of adenosine A_2A receptors by loop perfusion blunted the TGF response. Global NOS blockade, but not nNOS specific inhibition, prevented A_2A receptor-mediated blunting of TGF. This suggests that A_2A receptors blunted the TGF response by activation of eNOS in the JGA. Moreover, global blockade of NOS enhanced TGF responses more than selective inhibition of nNOS, indicating an additional role of eNOS in modulating TGF.

The TGF mechanism is a negative feedback loop that senses changes in luminal NaCl delivery and reabsorption at the macula densa in the JGA and adjusts the vascular tone of the afferent arteriole accordingly (44). Increased tubular flow rates are associated with increased adenosine formation in the JGA and activation of TGF (30, 37). In contrast to the vasculature of most other organs, both A₁ and A₂ receptors are widely expressed in preglomerular vessels (17, 22, 51, 53). Adenosine importantly contributes to the renal microvascular tone via activation of A₁ and A₂ receptors. Studies in isolated and perfused renal afferent arterioles have demonstrated a biphasic response to adenosine, with A₁-mediated contraction in the low concentration range and A_2A-mediated dilatation at high concentration (5, 7, 12, 22).

Adenosine, via activation of A₁ receptors, has been demonstrated to mediate the TGF response in both rats (8, 45) and mice (3, 46), whereas ANG II, NO, and superoxide are important modulators (9, 24, 33, 49, 56). Studies have also suggested that TGF signals are coupled to autoregulatory preglomerular vasoconstriction through ATP-mediated activation of purinergic P2X₁ receptors (14). The controversies regarding the role of adenosine or ATP as the signaling molecule have been debated during the last years and may be due to regional differences of the kidney (13, 42).

Given the dilatory function of A_2A receptors in the afferent arteriole, it is possible that this subtype of adenosine receptors might have functional significance in modulating TGF. In a recent study, we showed that intratubular administration of an adenosine A_2A receptor antagonist (ZM-241385) enhanced the TGF response (6). Even though ZM-241385 is a potent and highly selective A_2A receptor antagonist, the narrow window for receptor inhibition makes it difficult to exclude any contribution of A_2B receptors. Perfusion with a potent and selective adenosine A₁ receptor antagonist (PSB-36) was associated with a paradoxical reversed TGF response (6). This finding suggested activation of adenosine A₂ receptors alone and supported the finding that cumulative application of adenosine in A₁ receptor-deficient mice was associated with a monophasic dilatory response in renal afferent arterioles (22).

In the present study, we show that intratubular administration with a potent and highly selective adenosine A_2A receptor agonist (CGS-21680) attenuated the maximal TGF response. Previous micropuncture experiments have shown that adenosine analogs delivered via intraluminal perfusion may access the vascular receptor sites in the JGA through permeable junctional complexes and intracellular pathways (8, 19, 41, 45). A₂ receptors have not been identified in any nephron segments (53), and, to the best of our knowledge, there is no evidence for expression of adenosine receptors in the macula densa. However, several publications have shown expression, and a functional role, of adenosine receptors in the preglomerular vessels (29, 43, 53). Therefore, the vascular cells of the afferent arteriole were suggested to be the predominant site of action. The dose used for CGS-21680 may affect A₃ receptors; however, studies have shown that A₃ receptors are not expressed in any nephron segment (53) and do not appear to have a major role in regulation of renal excretory function under normal conditions (26). The mechanism responsible for A₂ receptor-mediated dilatation of afferent arterioles and its modulation of TGF is not clear, but release of NO has been suggested (5, 12, 23, 36, 47).

In vitro studies in microperfused efferent arterioles, with or without adherent tubular segments with macula densa, showed that preconstricted arterioles dilated in response to increased macula densa NaCl (38), and in response to A₂ receptor activation (1). A possible efferent arteriole TGF works in the opposite direction from that in afferent arterioles, thus amplifying changes in glomerular capillary pressure and glomerular filtration rate. Our results, however, indicate that the site of action of A_2A receptors in the regulation of TGF is predominantly the afferent arterioles, since activation of adenosine postglomerular A_2A receptors would be associated with a reduction in efferent arteriole resistance, a fall in glomerular capillary pressure, and an enhanced TGF response.

NO has been shown to modulate the TGF response by damping adenosine A₁ receptor-mediated contraction. In TGF regulation, nNOS in the macula densa is generally thought to be the primary source of NO (39, 48, 55, 57). Perfusion with the nonselective NOS inhibitor l-NAME or the selective nNOS inhibitor l-NPA had no effect on P_SF at 0 nl/min, supporting the notion that basal production of NO in renal afferent arterioles is very low during control conditions (31). In the present study, we show that both nonselective NOS inhibition and selective nNOS inhibition enhanced TGF. However, the sensitization of the response was more pronounced with l-NAME, suggesting that eNOS also may modulate the response. The concentration of l-NPA used in the present study was shown in a recent study to give maximal sensitization of TGF (2) and should only inhibit nNOS. 7-Nitroindazole (7-NI) has been widely used to study the contribution of nNOS in regulation of TGF; however, new knowledge shows nonselective properties of this NOS inhibitor. The much more selective nNOS inhibitor used in the present study may explain the discrepancies with previous studies where 7-NI and global NOS inhibitors had generally similar effects in enhancing TGF (4, 48). A previous in vitro study in rabbits suggested that l-NAME added into the tubular perfusate had no effect on the afferent arteriole and only acted on the macula densa cells (16). The various results in these studies might be explained by different experimental approaches, and also that a higher dose of l-NAME was used in the present study.

Studies have suggested that eNOS expressed in the thick ascending limb (TAL) may influence TGF. NO produced in this region of the nephron can act both as an autacoid agent by inhibiting NaCl reabsorption (35) or act as a paracrine agent (54). The former would increase electrolyte load to the macula densa, thus enhancing TGF, whereas diffusion of NO to the macula densa would attenuate the response. This suggests that TAL-derived NO potentially could have dual effects on TGF. Our finding of a much stronger TGF response with l-NAME perfusion cannot be explained by an autacoid action of NO. If eNOS-derived NO from TAL acts on the macula densa, inhibition of this pathway during perfusion with l-NAME could contribute to the enhanced TGF response. However, considering that the short half-life of NO limits its paracrine actions, and also that intratubular l-NAME pretreatment did not impact P_SF at baseline, we suggest that the modulating effects on TGF are predominantly due to effects on the afferent arteriole.

To study the possibility that A₂ receptor activation interacts with NOS isoforms, the effects of the A_2A receptor agonist on TGF were studied during nonselective NOS inhibition or nNOS inhibition. During simultaneous l-NAME perfusion, A_2A receptor-mediated dilatation was absent; however, during nNOS inhibition, the A_2A receptor agonist attenuated the TGF response. This indicates that A_2A receptor stimulation and reduced TGF response is linked to activation of a NOS isoform other than nNOS, presumably eNOS. In whole kidney preparations, Hansen and colleagues (12) showed that the steady-state effect of intravenous adenosine administration was vasodilatation, as evidenced by reduced renal vascular resistance and increased renal blood flow. This effect was largely mediated by A_2A receptor activation. The dilatory action of adenosine was abolished in eNOS knockout mice and in wild types given l-NAME, suggesting eNOS as the source of NO that mediated the effects of A_2A receptor activation (12). A potential role for A_2A receptor-mediated activation of eNOS and modulation of vascular tone has also been demonstrated in rat aorta (36) and carotid arteries from mice (47) and piglets (23).

The signaling pathways for A_2A receptor interaction with eNOS in the kidney are not well characterized. However, in rat aortic endothelium, A_2A-mediated NO release required extracellular Ca²⁺ and Ca²⁺-activated K⁺ channels (K_Ca) (36). The K⁺ efflux, resulting from A_2A-coupled K_Ca channels, facilitated Ca²⁺ influx and activation of eNOS. This process may be facilitated by protein kinase A phosphorylation of eNOS via A_2A receptor stimulation of adenylate cyclase and increasing cAMP. Future studies are required to characterize the mechanism for interaction between A_2A receptors and eNOS in regulation of renal arteriolar responses and TGF.

In conclusion, the present study demonstrates an important role of adenosine A_2A receptors in modulating the TGF response by counteracting A₁ receptor-mediated vasoconstriction. The A_2A receptor-mediated attenuation of TGF was linked to stimulation of eNOS. The underlying mechanisms remain to be further investigated, but activation of eNOS in the afferent arteriole is suggested. Moreover, our findings support previous findings of a role of nNOS in regulating TGF but do also demonstrate an additional role of eNOS in blunting the response.

GRANTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-072183 (W. J. Welch) and DK-49870, DK-036079, and DK-068686 (C. S. Wilcox and W. J. Welch), The Swedish Society of Medicical Research, The Wenner-Gren Foundation, The Swedish Society of Medicine, Magnus Bergvall Foundation. and funds from the George E. Schreiner Chair of Nephrology.

DISCLOSURES

None