Adenosine inhibits renin release from juxtaglomerular cells via an A₁ receptor-TRPC-mediated pathway

Abstract

Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A₁ receptor (A₁R) calcium-mediated pathway. How this occurs is unknown. In cardiomyocytes, adenosine increases intracellular calcium via transient receptor potential canonical (TRPC) channels. We hypothesized that adenosine inhibits renin release via A₁R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A₂R activation. Using primary cultures of isolated mouse JG cells, immunolabeling demonstrated renin and A₁R in JG cells, but not A₂R subtypes, although RT-PCR indicated the presence of mRNA of both A_2AR and A_2BR. Incubating JG cells with increasing concentrations of adenosine decreased renin release. Different concentrations of the adenosine receptor agonist N-ethylcarboxamide adenosine (NECA) did not change renin. Activating A₁R with 0.5 μM N6-cyclohexyladenosine (CHA) decreased basal renin release from 0.22 ± 0.05 to 0.14 ± 0.03 μg of angiotensin I generated per milliliter of sample per hour of incubation (AngI/ml/mg prot) (P < 0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35 ± 0.08 μg AngI/ml/mg prot; P < 0.01), and blocked renin inhibition by CHA (0.28 ± 0.06 μg AngI/ml/mg prot; P < 0. 005 vs. CHA alone). The intracellular calcium chelator BAPTA-AM increased renin release by 55%, and blocked the inhibitory effect of CHA. Repeating these experiments in JG cells from A₁R knockout mice using CHA or NECA demonstrated no effect on renin release. However, RT-PCR showed mRNA from TRPC isoforms 3 and 6 in isolated JG cells. Adding the TRPC blocker SKF-96365 reversed CHA-mediated inhibition of renin release. Thus A₁R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A₂ receptors do not regulate renin release.

renin is produced by, stored in, and released from juxtaglomerular (JG) cells located in the lamina media of the afferent arteriole at the entrance to the glomerulus (7, 31). Two main intracellular second-messenger systems are known to regulate renin secretion: stimulation of renin release by the cyclic nucleotide cAMP; and inhibition or renin secretion by increased intracellular calcium (1–3, 16, 25, 30).

Adenosine is a purine nucleoside catabolite of ATP. Adenosine's effects are mediated by the activation of four G protein-coupled receptor (GPCR) subtypes: A₁, A_2A, A_2B, and A₃ (13, 44). Adenosine inhibits renin secretion in vivo and in vitro (9, 37). It is considered that adenosine from the macula densa inhibits renin via A₁ receptor (A₁R) activation, although the presence of A₁R on JG cells has yet to be demonstrated. Mice without the A₁R have high basal plasma renin concentrations and diminished ability for high levels of NaCl to inhibit renin (64). Churchill and Churchill (15, 17), using rat cortical slices, also demonstrated that the adenosine receptor agonist N6-cyclohexyladenosine (CHA) inhibited renin release at submicromolar concentrations, but at higher micromolar concentrations it stimulated renin release. This dichotomous response was explained such that A₁R adenosine receptors mediated the inhibition of renin, but at higher concentrations, stimulation of renin secretion was due to activation of the A₂R subtype (17). Churchill and colleagues proposed that adenylyl cyclase activity is inhibited by A₁R but stimulated by A₂R subtype activation (61). However, the presence of A_2AR or A_2BR on JG cells has yet to be demonstrated, and the cellular mechanisms explaining this stimulation are not known.

Additionally, extracellular calcium chelation was found to diminish renin inhibition by CHA, suggesting that extracellular calcium is critical in mediating the A₁R effect (48). Whereas A₁R inhibition of renin is considered to be calcium-dependent, the pathway for this calcium entry is not known. Recently, it has been shown in chick cardiomyocytes that A₁R activation mediates proarrhythmic calcium entry through transient receptor potential canonical (TRPC) channel 3, functioning as receptor-operated channel (ROC) (62). TRPC channel proteins have been identified as downstream molecules in a GPCR signaling pathway, and are involved in a variety of cell functions due to their ability to regulate intracellular calcium signaling. TRPC isoforms 3, 6, and 7 have been described as being activated by agonists acting on a range of GPCRs such as A₁R, and to increase intracellular calcium concentration (26) in a mechanism known as receptor-operated channel entry (ROCE).

Because of these previous observations, we hypothesized that adenosine activation of A₁R inhibits renin release through a calcium-mediated pathway involving calcium entry through ROCE via TRPC channels. However, adenosine at higher concentrations should stimulate renin through A₂R activation.

MATERIALS AND METHODS

JG Cell Preparation

Isolation of mouse JG cells.

This study conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and our protocol was approved by the Institutional Animal Care and Use Committee of the Henry Ford Health System. In all of the following protocols, we used primary culture of isolated mouse JG cells. Male C57BL/6 mice (8–9 wk old) were killed, and JG cells were isolated following a protocol based on the methods of Della Bruna (21, 39) et al., which we modified to improve the harvest, purity, and stability of the primary culture as previously described in detail (45, 51, 52, 54). Some of the protocols also involved JG cells isolated from A₁R homozygous knockout mice (male and female) that were obtained from A₁AR heterozygous knockout breeders (C57BL/6 background) provided by Jürgen Schnermann (National Institute of Diabetes and Digestive and Kidney Diseases). The genotype was confirmed by tail-snip PCR. JG cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ in air. After 48 h of incubation, the culture medium was removed and replaced with 250 μl of fresh, prewarmed, serum-free culture medium containing 1.2 mM calcium, with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; Sigma, St Louis, MO) 0.1 mM dissolved in DMSO (Sigma), plus the various drugs to be tested as described below. JG cells were incubated for 2 h, after which the supernatant was collected, centrifuged to remove any cellular debris, and assayed for the activity of renin released into the medium (see below). All protocols used paired experiments run on pooled JG cells from the same harvest, where the preparation from the cortices of four mice were pooled and divided, and used separately for control and treatment in a given culture (n = 1) on a given day.

JG Cell Adenosine Receptor Expression

Immunolabeling of A₁R and renin in JG cells.

We placed primary cultures of JG cells on Poly-d-Lysine-coated cover slips for 48 h. The medium was then removed and the cells were fixed for 30 min with freshly prepared 4% paraformaldehyde diluted in PBS, then washed with Tris-buffered saline Tween (TBST) three times for 5 min each. The fixed cells were permeabilized with 0.2% Triton X-100 for 10 min, then washed. Nonspecific binding was blocked with 5% BSA for 30 min. The cells were incubated for 1 h with an A₁R antibody (Sigma) (37) diluted 1:25 in 5% BSA. Cells were then washed and incubated with a goat anti-rabbit antibody labeled with Alexa Fluor 568 fluorescent dye (Invitrogen) diluted 1:100 in 5% BSA for 1 h. After incubation with the secondary antibody, cells were again washed and then incubated for 1 h with a 1:25 dilution of an antibody against renin protein (sheep anti-mouse FITC-labeled; Innovative Research). Cells were again washed and the coverslips were mounted on slides with Fluoromount (Southern Biotech Associates). The preparations were examined by confocal microscopy (Visitech Confocal System), excited at 488 nm, emission measured at >500 nm to obtain images of the renin antibody, and exited at 568 nm; emission was measured again at >590 nm for the A₁R antibody. This protocol was repeated four times, and each time images of at least 20 cells were taken.

Immunolabeling of A_2AR and renin in JG cells.

Using the same protocol as described above, labeling was performed using an A_2AR antibody (Alpha Diagnostic, San Antonio, TX) (43) at a concentration of 30 μg/ml. As a positive control we used a mouse hepatocytes cell line (AML-12; ATCC, Manassas, VA) (12) between the second and third passage, grown on Poly-d-Lysine-coated coverslips for 48 h, then fixed with 4% paraformaldehyde.

Coimmunolabeling of A_2BR and renin in JG cells.

We followed the same protocol as described above, but with the A_2BR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (10) at a 1:50 dilution. As positive control, we used mouse a hepatocyte cell line (AML-12; ATCC) (12) as above.

RT-PCR for A_2AR and A_2BR in JG cells.

RT-PCR was used to determine expression of A₂R subtypes in a preparation of pipette-selected isolated mouse JG cells that minimizes possible non-JG cell contamination. Using the same preparation described elsewhere (52, 53), individual JG cells were selected under a 40× Fisher Scientific microscope with a 1-ml pipette. A suspension of mouse liver cells was used as a positive control (36). A no-template control was used as a negative control. Isolated JG cells were resuspended in 1 ml of Tri-reagent (Molecular Research Center, Cincinnati, OH). Likewise, mouse liver was homogenized in 1 ml Tri-reagent, and total RNA was prepared according to the manufacturer's instructions. One microgram of extracted total RNA was reverse transcribed at 37°C using Omniscript reverse transcriptase (Qiagen, Valencia, CA), and 2 μl of mixture was taken for subsequent PCR. The following primers were used to detect the A_2AR (20): sense, 5′-cctgaattccactccggtaca-3′; antisense, 5′-cagttgttccagcccagcat-3′. The following primers were used to detect the A_2BR (20): sense, 5′-tcttcctcgcctgcttcgt-3′; antisense, 5′-ccagtgaccaaacctttatacctga-3′. Primer pairs for each transcript were designed to span an exon-intron-exon boundary (4). PCR was performed under the following conditions: 94°C for 2 min; 40 cycles at 95°C for 30 s, 60°C for 40 s, 72°C for 1 min, followed by a final extension at 72°C for 5 min. The reaction products were then held at 4°C. PCR products were run on a 2% agarose gel in 1× TBE. A_2AR and A_2BR give PCR products at 120 and 121 bp, respectively (20).

RT-PCR for TRPC 3, 6, and 7 in JG cells.

RT-PCR was used to examine the expression of the TRPC3/6/7 isoforms using pipette-selected isolated mouse JG cells. A no-template control was used as a negative control. Isolated JG cells were resuspended in 1 ml of Tri-reagent and total RNA was prepared according to the manufacturer's instructions. One microgram of extracted total RNA was reverse transcribed at 37°C using Omniscript reverse transcriptase (Qiagen) and 2 μl of mixture was taken for subsequent PCR. The following primers were used to detect TRPC3: sense, 5′-aagcccagtattaatgtcactg-3′; antisense, 5′-aaaaggcaaatgataatgacag-3′. The following primers were used to detect TRPC6: sense, 5′-agaccgttcatgaagtttgtag-3′; antisense, 5′-ttctttacattcagcccatatc-3′. The following primers were used to detect TRPC7: sense, 5′-agaatcaagatgtgcctcatag-3′; antisense, 5′-cattgacttcatcgttctctct-3′. PCR was performed under the following conditions: 94°C for 2 min; 40 cycles at 95°C for 30 s, 60°C for 40 s, 72°C for 1 min, followed by a final extension at 72°C for 5 min. The reaction products were then held at 4°C. PCR products were run on a 2% agarose gel in 1× Tris (0.89 M) plus borate (0.09 M) and EDTA (0.02 M); TBE. TRPC3, TRPC6, and TRPC7 give PCR products at 224, 225, and 247 bp, respectively (71).

Renin Release Protocols

Protocols using JG cells isolated from C57 wild-type mice.

CONCENTRATION-DEPENDENT RESPONSE OF RENIN TO ADENOSINE RECEPTOR STIMULATION WITH ADENOSINE WITH OR WITHOUT THE ADENOSINE DEAMINASE INHIBITOR (N = 8).

We tested the possible concentration-dependent, biphasic effect of adenosine (Sigma) on renin release. Primary cultures of JG cells were incubated with DMEM and either 1) vehicle, 2) 0.1 μM adenosine (40), 3) 1 μM adenosine (64), or 4) 10 μM adenosine (40). Cells were incubated for 2 h, after which the medium was collected for determination of renin release and the cells were harvested for determination of total JG protein.

In a different set of experiments (n = 8) we preincubated JG cells with an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; Sigma) 50 μM (42) for 1 h to block adenosine degradation. Afterward, JG cells were incubated with the different concentrations (0.1 μM, 1 μM, or 10 μM) of adenosine alone for 2 h, after which the medium was collected for determination of renin release and the cells were harvested for determination of total JG protein.

CONCENTRATION-DEPENDENT RESPONSE OF RENIN TO ADENOSINE A₂R STIMULATION WITH NECA (N = 8).

We tested possible concentration-dependent effects of activation of the adenosine A₂R on renin release using the adenosine receptor agonist N-ethylcarboxamide adenosine (NECA; Sigma) (18, 57, 65). Primary cultures of JG cells were incubated with DMEM and either 1) vehicle, 2) 0.1 μM NECA (40), 3) 3 μM NECA (65), or 4) 10 μM NECA (18). Cells were incubated for 2 h, after which the medium was collected for determination of renin release and the cells were harvested for determination of total JG protein.

CONCENTRATION-DEPENDENT RESPONSE OF RENIN TO ADENOSINE RECEPTOR STIMULATION WITH CHA (N = 11).

We tested the reported biphasic (18) concentration-dependent effect of the adenosine receptor agonist CHA (Sigma) on renin release on the basis of the original protocol described by Churchill and Churchill (17). Primary cultures of JG cells were incubated with DMEM and either 1) vehicle, 2) 0.5 μM CHA, 3) 5 μM CHA, or 4) 50 μM CHA. Cells were incubated for 2 h, after which the medium was collected for determination of renin release and the cells were harvested for determination of total JG protein.

STIMULATION OF THE ADENOSINE RECEPTOR WITH CHA IN THE ABSENCE OF EXTRACELLULAR CALCIUM (N = 10).

To test whether the presence of extracellular calcium is required for CHA to inhibit renin release, we used EGTA (Sigma) to chelate the extracellular calcium concentration nominally to zero. Primary cultures of JG cells were incubated with DMEM and either 1) normal control, 2) 0.5 μM CHA, 3) 2 mM EGTA, or 4) EGTA plus 0.5 μM CHA. Cells were incubated for 2 h, after which the medium was collected for determination of renin release and the cells were harvested for determination of total JG protein.

STIMULATION OF THE ADENOSINE RECEPTOR WITH CHA AFTER CLAMPING INTRACELLULAR CALCIUM (N = 14).

To show that increased intracellular calcium is necessary for renin inhibition by CHA, we used the intracellular calcium chelator BAPTA-AM (Biomol) (51, 52) in the presence or absence of the adenosine receptor agonist CHA. Isolated JG cells were treated with either 1) vehicle, 2) 0.5 μM CHA, 3) 100 μM BAPTA-AM, or 4) BAPTA plus 0.5 μM CHA. Cells were incubated for 2 h, after which the medium was collected for determination of renin release and the cells were harvested for determination of total JG protein.

STIMULATION OF THE ADENOSINE RECEPTOR WITH CHA AFTER TRPC CHANNEL BLOCKADE (N = 8).

To determine whether the adenosine-mediated entry of calcium was via the TRPC channel in JG cells, we used the TRPC blocker SKF-96365 (SKF; Enzo Life Biosciences, NY) (23, 32, 34) in the presence and absence of the adenosine A₁R agonist CHA. Isolated JG cells were treated with either 1) vehicle, 2) 50 μM SKF (32, 34), 3) 0.5 μM CHA, or 4) SKF plus CHA. Cells were incubated for 2 h, after which the medium was collected for determination of renin release and then the cells were harvested for determination of total JG protein.

Protocols using JG cell isolated from A₁R knockout (KO) mice.

STIMULATION OF THE ADENOSINE RECEPTOR WITH CHA IN A₁R KO MICE (N = 5).

To test whether removing the A₁R, and therefore the possible renin-inhibiting influence of that receptor, would allow A₂R signaling to be unmasked, we used isolated JG cells obtained from kidneys harvested from A₁R KO mice (14, 72). First, to show the absence of an A₁R, we employed a high concentration of CHA (100 μM). JG cells were treated with either 1) vehicle or 2) 100 μM CHA. Cells were incubated for 2 h, after which the medium was collected for determination of renin release and the cells were harvested for determination of total JG protein.

STIMULATION OF THE A₂R WITH NECA IN A₁R KO MICE (N = 5).

To test whether direct stimulation of A₂R in the absence of A₁R would unmask the proposed A₂R-mediated stimulation of renin, we used a high concentration (100 μM) of the selective A₂R agonist NECA in the absence of A₁R. Isolated JG cells obtained from kidneys harvested from A₁R KO mice were treated with either 1) vehicle or 2) 100 μM NECA. Cells were incubated for 2 h, after which the medium was collected for determination of renin release and the cells were harvested for determination of total JG protein.

Assays

Renin release.

After 2 h of JG cell incubation, the medium was drawn off, centrifuged, and the supernatant was recovered for assay of renin concentration (ANG I generation). Samples were incubated for 3 h with excess rat angiotensinogen as substrate, as previously described (6, 45, 50). Renin consumed less than 15% of exogenous substrate to ensure the enzymatic reaction remained in first-order kinetics. ANG I generation was assayed using a Gamma Coat RIA kit (DiaSorin, Stillwater, MN) as previously described (5, 45, 50–53). Values for renin concentration (micrograms of ANG I generated per milliliter of sample per hour of incubation) were corrected for JG cell total protein and are presented hereafter as μg AngI/ml/h/mg prot.

Protein concentration.

The protein concentration in JG cell lysates used to correct renin release per milligram of protein was determined using the Coomassie plus Protein Assay Reagent kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions as previously described (50, 52).

Statistical Analysis

All data were derived and analyzed from paired control and experimental permutations from a pooled primary culture obtained on a single day (n = 1) from the cortices of four mice. Changes in renin release (as determined from ANG I production) compared with controls were evaluated using either a paired t-test or ANOVA for repeated measures with a Bonferoni post hoc test. We considered a corrected value of P < 0.05 to be significant. In the figures, for the sake of simplicity, all statistically significant changes are represented as P < 0.05, whereas the actual P values are presented in Results. To normalize and simplify data presentation, Figures 6–Figures 13 show renin release from JG cells as a change expressed as a percent of control. However, actual data are provided in the Results narrative.

Fig. 6.

Renin release from JG cells under basal conditions (control) or after incubation with three different concentrations of adenosine (0.1 μM, 1 μM, and 10 μM) in the presence (black bars) or absence (gray bars) of the adenosine deaminase inhibitor EHNA. *P < 0.05 vs. control.

Fig. 13.

Renin release from JG cells isolated from A₁R KO mice under basal conditions (cntrol) or after incubation with 100 μM of the adenosine receptor agonist NECA. NECA did not significantly change renin release.

RESULTS

Coimmunolabeling of A₁R and Renin in JG Cells

We used an A₁R antibody with immunolabeling and confocal microscopy, and found positive labeling for the A₁R isoform in JG cells grown on coverslips. Figure 1 shows a typical example in which the A₁R isoform (shown in red) is localized in a single JG cell, as identified by positive labeling for renin (shown in green). The merged image shows the same JG cell labeled with both antibodies (yellow).

Fig. 1.

Immunoflourescence and confocal microscopy of a single JG cell using two antibodies, one specific for renin (in green) to confirm this is a JG cell, and another specific for the A₁R isoform (in red). The third panel shows both renin and A₁R in the same JG cell (merged image).

Coimmunolabeling of A_2AR or A_2BR and Renin in JG Cells

We used an A_2AR antibody with immunolabeling and confocal microscopy to identify the A_2AR isoform. However, we did not find the A_2AR isoform expressed in JG cells (Fig. 2), which stained positively for renin (shown in green). The same antibody showed expression of the A_2AR isoform in the mouse hepatocytes cell line (AML-12) used as positive control (12) (shown in red).

Fig. 2.

Immunoflourescence and confocal microscopy using a specific antibody for the A_2AR isoform (in red). JG cells labeled positive for renin (in green) did not express A_2AR, whereas A_2AR was expressed in the positive control AML-12 culture (in red).

Similarly, the same protocol was performed with an A_2BR selective antibody and found no labeling for A_2BR in the renin-positive JG cells, but did find positive labeling in the AML-12-positive controls (12) (Fig. 3). Thus we were not able to detect protein for either A₂R subtype isoform in mouse JG cells using immunolabeling and confocal microscopy.

Fig. 3.

Immunoflourescence and confocal microscopy using a specific antibody for the A_2BR isoform (in red). JG cells labeled positive for renin (in green) did not express A_2BR, whereas A_2BR was expressed in the positive control AML-12 culture (in red).

RT-PCR for A_2AR and A_2BR in JG Cells

Figure 4A shows that RT-PCR performed on 1 μg of total RNA from JG cells gave a product at the expected size of 120 bp in both isolated JG cells and in the positive control of mouse liver. The no-template negative control showed no amplification. These results suggest that there is expression of A_2AR in JG cells. Figure 4B shows that RT-PCR performed on 1 μg of total RNA from JG cells gave a product at the expected size of 121 bp in both isolated JG cells and in the positive control of mouse liver. The no-template negative control showed no amplification. These results suggest that there is expression of A_2BR in JG cells.

Fig. 4.

A: RT-PCR for A_2AR identifying a band at a 120 bp. Column 1 is total RNA obtained from the positive control of liver, column 2 is total mRNA obtained from isolated JG cells, column 3 is the no-template negative control, and column 4 is the calibration scale (100 bp ladder). B: RT-PCR for A_2BR identifying a band at a 121 bp. Column 1 is total RNA obtained from the positive control of liver, columns 2 and 3 represent total mRNA obtained from isolated JG cells, column 4 is the no-template negative control, and column 5 is the calibration scale (100 bp ladder).

RT-PCR for TRPC3/6/7

Figure 5 shows that the RT-PCR performed on 1 μg of total RNA gave a positive product for TRPC3 and TRPC6 at the expected size of 224 and 225 bp, respectively. However, no product was evident at 247 bp corresponding to TRPC 7. The no-template negative control showed no amplification. These results suggest that there is expression of TRPC3 and TRPC6. Furthermore, the data suggest the TRPC6 isoform is more abundant than TRPC3 because amplification was performed on the same cDNA. It also appears that there is no TRPC 7 in JG cells.

Fig. 5.

Two different gels (one for TRPC3 and one for TRPC6 and TRPC7) were run under the same conditions. Shown is a composite of both gels. RT-PCR for TRPCs 3, 6, and 7 identifying two bands at 224 and 225 bp. Columns labeled for TRPC3, 6, and 8 are total mRNA obtained from isolated JG cells. The next (unlabeled) column is the no-template negative control; and the far right column is the calibration scale (100 bp ladder).

Concentration-Dependent Response of Renin to Adenosine Receptor Stimulation with Adenosine, with or Without the Adenosine Deaminase Inhibitor

Incubation of JG cells with 0.1 μM adenosine decreased basal renin release by 40%, from 1.66 ± 0.49 to 0.99 ± 0.32 μg AngI/ml/h/mg prot (P < 0.005 vs. untreated control) (Fig. 6). Incubation of JG cells with 1 μM adenosine likewise decreased renin release to 1.09 ± 0.37 μg AngI/ml/h/mg prot (P < 0.025 vs. control). Using 10 μM adenosine also decreased renin release to 1.49 ± 0.47 μg AngI/ml/h/mg prot. The lower concentration of adenosine seemed to be more effective at inhibiting renin release than the higher concentration.

After 1 h of pretreatment with EHNA, incubation of JG cells with 0.1 μM adenosine decreased basal renin release by 50%, from 1.3 ± 0.35 to 0.62 ± 0.21 μg AngI/ml/h/mg prot (P < 0.001 vs. untreated control). Incubation of JG cells with 1 μM adenosine similarly decreased renin release to 0.77 ± 0.26 μg AngI/ml/h/mg prot (P < 0.025 vs. control). Using 10 μM adenosine also decreased renin release to 0.96 ± 0.31 μg AngI/ml/h/mg prot (P < 0.005 vs. control). Responses were not concentration-dependent because all concentrations of adenosine with EHNA-inhibited renin.

Concentration-Dependent Response of Renin to Adenosine Receptor Stimulation with NECA

Renin release did not change when JG cells were incubated with 0.1 μM NECA (1.49 ± 0.24 and 1.11 ± 0.21 μg AngI/ml/h/mg prot, respectively; Fig. 7). Renin release remained unchanged using 3 μM or 10 μM NECA (1.03 ± 0.2 μg AngI/ml/h/mg prot and 1.21 ± 0.19 μg AngI/ml/h/mg prot, respectively). Thus adenosine receptor stimulation with NECA did not increase renin release at any concentration.

Fig. 7.

Renin release from JG cells under basal conditions (control) or after incubation with different concentrations (0.1 μM, 3 μM, and 10 μM) of the adenosine receptor agonist NECA. NECA did not significantly change renin release at any concentrations. *P < 0.05 vs. control.

Concentration-Dependent Response of Renin to Stimulation of the Adenosine Receptors Using CHA

Incubation of JG cells with 0.5 μM CHA decreased basal renin release by 50%, from 0.40 ± 0.05 to 0.20 ± 0.03 μg AngI/ml/h/mg prot (P < 0.01 vs. untreated control) (Fig. 8). Renin release was similarly suppressed with either 5 μM or 50 μM CHA to 0.17 ± 0.02 μg AngI/ml/h/mg prot (P < 0.05 vs. control) and 0.23 ± 0.04 μg AngI/ml/h/mg prot (P < 0.01 vs. control). Thus in contrast to our hypothesis, all concentrations of CHA that we used inhibited renin similarly.

Fig. 8.

Renin release from JG cells under basal conditions (control) or after incubation with different concentrations (0.5 μM, 5 μM, and 50 μM) of the adenosine receptor agonist CHA. CHA similarly and significantly decreased renin release at all submicromolar and micromolar concentrations. *P < 0.05 vs. control.

Stimulation of the Adenosine Receptor with CHA in the Absence of Extracellular Calcium

The adenosine receptor agonist CHA (0.5 μM) decreased renin release by 40% compared with control, from 0.22 ± 0.05 to 0.14 ± 0.03 μg AngI/ml/h/mg prot (P < 0.025) (Fig. 9). Incubation of JG cells with the extracellular calcium chelator EGTA increased basal renin release by 60% to 0.35 ± 0.08 μg AngI/ml/h/mg prot (P < 0.002). When JG cells were incubated with both CHA and EGTA, renin release was increased twofold, to 0.28 ± 0.06 μg AngI/ml/h/mg prot compared with treatment with CHA alone (P < 0.005). Thus extracellular calcium-chelation blocked the inhibition of renin by CHA.

Fig. 9.

Renin release from JG cells under basal conditions (control) or after incubation with the adenosine receptor agonist CHA (0.5 μM). CHA significantly decreased renin release, whereas the extracellular calcium chelator EGTA (2 mM) increased renin release. Incubation with both EGTA and CHA completely reversed the CHA inhibition of renin. *P < 0.05 vs. control. #P < 0.05 vs. CHA alone.

Stimulation of the Adenosine Receptor with CHA After Clamping Intracellular Calcium

The adenosine receptor agonist CHA alone decreased renin release by 35%, from 0.19 ± 0.02 to 0.12 ± 0.02 μg AngI/ml/h/mg prot (P < 0.025 vs. control) as expected (Fig. 10). Incubation of JG cells with the intracellular calcium chelator BAPTA-AM increased renin release by 55% from control as expected (53), to 0.28 ± 0.05 μg AngI/ml/h/mg prot (P < 0.001 vs. control). When JG cells were incubated with both CHA plus BAPTA-AM, renin release increased to 0.25 ± 0.05 μg AngI/ml/h/mg prot (P < 0.025 vs. control and P < 0.02 vs. CHA alone). These results were not different from JG cells treated with BAPTA-AM alone. Thus clamping intracellular calcium concentration completely blocked CHA-mediated inhibition of renin release.

Fig. 10.

Renin release from JG cells under basal conditions (control) or after incubation with the adenosine receptor agonist CHA (0.5 μM). CHA significantly decreased renin release. The intracellular calcium chelator BAPTA-AM (1 μM) significantly increased renin release, and incubation with both BAPTA and CHA returned renin release to values similar to BAPTA alone, completely reversing the CHA inhibition of renin. *P < 0.05 vs. control. #P < 0.05 vs. CHA alone.

Stimulation of the Adenosine Receptor with CHA After TRPC Channel Blockade

The adenosine receptor agonist CHA alone decreased renin release by 40%, from 0.82 ± 0.2 to 0.45 ± 0.09 μg AngI/ml/h/mg prot (P < 0.009 vs. control) (Fig. 11). Incubation of JG cells with the TRPC channel blocker SKF did not changed basal renin release (0.76 ± 0.14 μg AngI/ml/h/mg prot). However, when JG cells were incubated with both CHA plus SKF, renin release returned to basal, to 1.02 ± 0.19 μg AngI/ml/h/mg prot (P < 0.004 vs. CHA alone), a result not different from JG cells treated with SKF alone. Thus blocking calcium entry via TRPC channels reversed the CHA-mediated inhibition of renin release.

Fig. 11.

Renin release from JG cells under basal conditions (control) or after incubation with the adenosine receptor agonist CHA (0.5 μM). The TRPC channel blocker SKF (50 μM) alone did not change basal renin release. CHA significantly decreased renin release, and incubation with both SKF and CHA returned renin release to values similar to control values, completely reversing the CHA inhibition of renin. *P < 0.05 vs. control. #P < 0.05 vs. CHA alone.

Stimulation of the Adenosine Receptor with CHA in A₁R KO Mice

Basal renin release did not change when JG cells were incubated with the adenosine receptor agonist CHA (100 μM), from 5.30 ± 0.71 to 5.25 ± 0.78 μg AngI/ml/h/mg prot (Fig. 12). Thus A₂R stimulation with CHA did not change renin release in JG cells from A₁R KO mice.

Fig. 12.

Renin release from JG cells isolated from A₁R KO mice under basal conditions (control) or after incubation with 100 μM of the adenosine receptor agonist CHA. CHA did not significantly change renin release.

Stimulation of the Adenosine Receptor with NECA in A₁R KO Mice

Basal renin release did not change when JG cells were incubated with the adenosine receptor agonist NECA (100 μM), from 2.58 ± 0.66 to 2.28 ± 0.36 μg AngI/ml/h/mg prot in the absence of A₁R (Fig. 13). Thus NECA did not change renin release in JG cells from A₁R KO mice.

DISCUSSION

The initial aim of this work was to study the cellular mechanisms by which adenosine acts through a reported biphasic effect on renin release (47); inhibiting via A₁R activation and low (submicromolar) concentrations, but stimulating via A₂R at higher (micromolar) concentrations of adenosine. We hypothesized that adenosine activation of the A₁R inhibits renin release through a calcium-mediated receptor-operated pathway involving TRPC channels. However, adenosine at higher concentrations should stimulate renin through A₂R activation (47), presumably via some cAMP-mediated stimulation. First, we showed (for the first time) that A₁R is expressed in JG cells, and its activation leads to inhibition of renin release. We also found this inhibition occurs through a calcium-mediated pathway, because when we removed the extracellular calcium or clamped intracellular calcium by chelation, we eliminated the inhibitory effect of A₁R activation. Additionally, we found that the A₁R-mediated influx of calcium can be attributed to TRPC channels. When we tested for ROCE-associated TRPC expression, we found TRPC 3 and TRPC 6, but not TRPC 7, and furthermore, TRPC6 appears to be more abundant, and therefore the more likely candidate to mediate in the adenosine-mediated calcium influx.

Adenosine is a purine nucleoside catabolite of ATP. Adenosine's effects are mediated by the activation of four GPCR subtypes: A1, A_2A, A_2B, and A₃. Many cell types express multiple adenosine receptor subtypes, but in some cell types activation of these receptors exert few effects, whereas in others the same receptors produce profound effects (44). The A₁ and A_2A subtypes are high-affinity receptors, whereas A_2A and A₃ receptors are low-affinity receptors. A₁R is a 36-kDa protein that couples primarily to Gi. A₁R activation decreases cAMP in adipocytes and increases intracellular calcium in smooth muscle cells (41). A_2AR is a 45- to 55-kDa protein that couples to Gs. Activation of the A_2AR stimulates adenylyl cyclase activity, resulting in increased intracellular cAMP levels. A_2BR is a 37-kDa GPCR that couples to Gs/Gq and is a low-affinity receptor. Activation of the A_2BR also stimulates adenylyl cyclase activity, resulting in increased intracellular cAMP levels (44).

Adenosine is known to be involved in the mediation of the macula densa-dependent inhibition of renin release in the presence of high tubular NaCl, as part of a mechanism recognized as tubuloglomerular feedback (33, 48, 55, 69). The inhibition of renin release by adenosine or by activation of the A₁R has been reported in vivo (66, 67). When adenosine was infused into the renal artery of canine kidneys, it blunted renin secretion (22). Additionally, in A₁R-deficient mice the plasma renin concentration was significantly elevated (64). This effect is consistent with the results presented in the present study. We also observed that renin release from JG cells isolated from A₁R KO mice was higher than that observed from JG cells from wild-type mice, although this effect was not observed in a report by Kim et al. (35), who observed no change in renin release in conscious A₁R KO mice compared with wild-type mice. In various in vitro studies, adenosine analogs inhibit renin release from rabbit renal cortical slices (8) or rat cortical slices (61), suggesting that adenosine inhibits renin release via A₁R activation. However, a study in transgenic mice in which A₁R expression was augmented in vascular smooth muscle and had enhanced tubuloglomerular feedback; the basal plasma renin concentrations were not changed, and neither was the suppression of renin secretion after volume expansion (49). Interestingly, although there is considerable indirect evidence for A₁R in JG cells, the actual presence of the A₁R has not previously been shown. In the present study, we confirm that existing bias that JG cells do contain the A₁R.

In vascular smooth muscle cells, A₁R mediates vasoconstriction by increasing intracellular calcium (29). The A₁R is a G_i-coupled receptor whose binding leads to activation of phospholipase C, increasing intracellular calcium (28, 29). Because JG cells appear in the afferent arteriole (45, 60, 70) contiguous to the vascular smooth muscle, it is likely that A₁R activation also increases intracellular calcium in JG cells, inhibiting cAMP formation and blunting renin release (50–53). This is consistent with our finding that JG cell A₁R activation inhibits renin release through a calcium channel-mediated pathway, in our demonstration that increases in both extracellular and intracellular calcium were required for the A₁R inhibition of renin.

In chick cardiomyocytes, adenosine-induced calcium entry via A₁R activation was shown to be mediated by the TRPC3 channel (62). Likewise, calcium entry in afferent arteriolar smooth muscle also involved activation of TRPC channels (24). Because of the coupling of TRPC-mediated calcium entry in related contractile tissues, we proposed that adenosine activation of A₁R in JG cells could also be mediated by TRPC channels. The TRPC family includes seven isoforms (TRPC1 to TRPC7) that have been divided into two general subfamilies on the basis of structural and functional similarities: TRPC1/4/5 and TRPC3/6/7 (19, 26). TRPC channels can be homomeric or heteromeric assemblies between four TRPC subunits. Even more interesting, oligomerization can occur within and between subfamilies or even beyond a given TRPC family altogether, which may generate highly distinct channels in different cell types (32). TRPC channels have been identified into two groups on the basis of whether they operate as store-operated channels (SOCs), including TRPC1/4/5, or as receptor-operated channels (ROCs), including isoforms TRPC3/6/7. SOCs act by emptying calcium from intracellular stores. ROCs act via activation of phospholipase C-mediated diacylglycerol production following stimulation of GPCRs (26, 58, 59). ROCs are not dependent on the state of intracellular calcium stores, but are regulated by receptor occupation and diacylglycerol formation. Thus we searched for mRNA expression of the TRPC associated with receptor-operated calcium entry, including isoforms TRPC3, 6, and 7 by RT-PCR. We found that JG cells did not contain isoform 7. We did find mRNA expression for both isoforms 3 and 6, and isoform 6 appears to be in greater abundance. Although not definitive, this suggests that isoform 6 is the likely candidate to mediate A₁R-ROC calcium entry.

To test the involvement of TRPC in the A₁R-calcium mediated-inhibition of renin release, we used a nonselective TRPC inhibitor, SKF-96365 (23, 32), in a concentration reported to produce an affect in U-87 MG cells (a malignant glioma cell line) (68) and in an MDCK cell line (34) in which concentration-response studies were performed. We found that TRPC channel inhibition completely reversed CHA-mediated decreased renin release. Thus we conclude that A₁R activation of JG cells results in TRPC channel-mediated calcium entry and the resulting calcium-mediated inhibition of renin release, and that this is likely via TRPC6 (see above).

The role of adenosine A₂R subtype activation on renin release is less clear because several different outcomes have been reported. Using rat renal cortical slices with the adenosine receptor agonist CHA at a submicromolar concentration, Churchill and Churchill (15, 17) reported inhibition of renin release, whereas at high micromolar concentration CHA stimulated renin release. This latter effect was attributed to A₂R activation, without directly being shown. Pfeifer et al. (56) used an A₂R antagonist in vivo in rats and reported no involvement of the A₂R in regulation of adenosine-mediated renin release, although another in vivo study in dogs showed that a selective A₂R agonist stimulated renin release (46).

Incubation of our isolated mouse JG cells with different concentrations of adenosine uniformly inhibited renin release, but we did not find any indication of an increase of renin at higher concentrations. When the same experiments were repeated in the presence of the adenosine deaminase inhibitor to guarantee adenosine activation of the receptors, there was still no increase in renin. Additionally, the (nonselective) adenosine receptor agonist NECA, with a high affinity for the A₂R at different concentrations, did not increase renin release. This result was unexpected because the concentrations we used were selected to mimic those by Churchill and Churchill (16, 17), and high concentrations were expected to increase renin release via activation of the A₂R. Consistent with this, when we repeated the Churchill et al. (18) dose-response experiment with CHA with our isolated mouse JG cells, we found decreased renin release at submicromolar concentrations, and we maintained this inhibition of renin using a high micromolar concentration. Thus, we were unable to repeat those findings. Our results are consistent with similar data from Kurtz et al. (39), who previously reported that NECA had no effect on renin release. The concentration of NECA selected for the above experiments to stimulate A₂ receptor subtypes has been reported before (17, 38, 65). The response to all of the different concentrations was the same; renin remained unchanged.

Importantly, despite data for and against a role for A₂R in adenosine, no one has ever shown that either receptor subtype existed on JG cells. Thus we attempted to resolve whether they exist on JG cells. In the present study, we found mRNA for both A_2AR and A_2BR using RT-PCR. However, we found no direct evidence that isolated mouse JG cells express either A_2AR or A_2BR protein. Our attempts to show immunostaining of the receptors showed A₁R, but they did not identify either A₂R subtype. To ensure that our A₂R subtype antibodies would work for immunolabeling, we used a mouse hepatocytes cell line (AML-12) (12, 36) as a positive control because these are known to express both A₂R subtypes. These cells did demonstrate positive immunolabeling. We have also found that these antibodies work well in labeling A₂R subtypes in a human coronary artery endothelial cell line (unpublished observations). In the present study, we found positive labeling for A_2AR and A_2BR was achieved in AML-12 cells, whereas no labeling for either A_2AR or A_2BR was apparent in our renin-positive mouse JG cells, consistent with our negative functional data.

It is possible that our results regarding the absence of A₂R subtypes vary from previous reports due to a species difference (mouse vs. rat), or it could be that our isolated JG cells are not influenced by indirect non-JG cell interactions inherent in rat cortical slices or other previous whole kidney studies (17, 46). Other regulatory factors influencing renin release indirectly but related to adenosine in the whole kidney may not be present in a pure JG cell preparation. The formation of adenosine is not limited to release from MD cells. It is possible that the bulk of the ATP present in vivo is locally hydrolyzed in the extracellular space to form adenosine, resulting in various actions (11). It is of course possible that the antibodies work in some cell types (hepatocytes) but not others (JG cells).

Several studies describe a cross-talk between adenosine A₁R and A₂R subtypes in the mouse myocardium. Both A_2AR and A_2BR are necessary for A₁R-mediated myocardial protection (72). Thus we speculated that perhaps the effect of A₂R activation was masked by the presence of A₁R or the inhibitory response of A₁R activation. To this end, we decided to use JG cells from A₁R KO mice to test whether the absence of A₁R or A₂R had some effect on renin release. Basal renin release was higher in A₁R KO mice compared with the control from experiments performed with wild-type mice, but this was expected because in A₁R-deficient mice the plasma renin concentration has been reported to be significantly elevated (56). When JG cells isolated from A₁R KO mice were incubated with a high concentration of CHA, renin release remained completely unchanged compared with control. This confirmed the functional absence of A₁R. Likewise, when JG cells isolated from A₁R KO mice were incubated with a high concentration of NECA, which has a high affinity for A₂R, renin release again remained unchanged. We did not find that higher concentrations of either CHA or NECA stimulated renin release, as had been previously reported. Renin either remained unchanged or it decreased.

Although we found evidence for A_2AR and A_2BR mRNA in JG cells by RT-PCR, we could not find functional evidence to confirm the expression of A_2AR/A_2BR protein in JG cells. The majority of our results indicate that neither adenosine type 2 receptor exerts any direct effects on JG cell renin secretion in wild-type or in A₁R KO mice, undermining the biphasic hypothesis (17).

In summary, we found that JG cells express A₁R, and that its activation leads to a calcium-mediated inhibition of renin release, presumably via the calcium inhibitable isoform(s) of adenylyl cyclase as previously described (27, 53). Our data suggest that A₁R activation involves participation of TRPC channels, probably operating as SOCs (62) through TRPC6. We did not find any direct or indirect evidence of A₂R subtypes functioning in the control of renin release in mouse JG cells, and we were unable to demonstrate any biphasic or renin-stimulating effect of adenosine as had been previously reported (17). Thus our studies suggest that JG cell renin secretion responds to adenosine only through an A₁R-mediated signaling pathway involving TRPC-mediated calcium entry, which inhibits renin release.

Perspectives

Elevated blood pressure and increased circulating volume inhibit renin secretion via negative feedback. It is believed that adenosine and the A₁ receptor play crucial roles in regulating this negative feedback response. Elevated blood pressure (64) and volume expansion (35) are unable to lower renin secretion in A₁R KO mice. However, how adenosine specifically transduces changes in renin secretion in response to changes in blood pressure or volume status has remained elusive. The data presented in this manuscript offer a novel answer. It is known that renin secretion is inversely related to intracellular calcium levels (52), and intracellular calcium inhibits renin secretion by decreasing intracellular cAMP levels via the deactivation of calcium-sensitive adenylate cyclases (52) and the activation of calcium-sensitive phosphodiesterases (50). The data from this manuscript suggest that TRPC channels are the mechanism coupled to A₁R by which adenosine increases intracellular calcium to inhibit renin secretion from the JG cell. This finding is consistent with additional literature implicating calcium as the second messenger by which adenosine, and by extension pressure, volume, and distal NaCl delivery, regulate changes in renin secretion.

GRANTS

This work was supported by funding from National Heart, Lung, and Blood Institute Grant PPG 5PO-1HL-090550-03.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: M.C.O.-C., D.K.A., P.H., R.D.L., and W.H.B. conception and design of research; M.C.O.-C. and P.H. performed experiments; M.C.O.-C., D.K.A., P.H., R.D.L., and W.H.B. analyzed data; M.C.O.-C., D.K.A., P.H., R.D.L., and W.H.B. interpreted results of experiments; M.C.O.-C. and W.H.B. prepared figures; M.C.O.-C., D.K.A., P.H., and W.H.B. drafted manuscript; M.C.O.-C., D.K.A., P.H., R.D.L., and W.H.B. edited and revised manuscript; M.C.O.-C., D.K.A., P.H., R.D.L., and W.H.B. approved final version of manuscript.