Adenylyl Cyclase Isoform V Mediates Renin Release From Juxtaglomerular Cells
We have shown previously that decreasing intracellular calcium in the juxtaglomerular cells increases both cAMP formation and renin release. We hypothesized that this is because of an interaction between intracellular calcium and the calcium-inhibitable isoform of adenylyl cyclase, type-V. We used primary cultures of juxtaglomerular cells isolated from C-57/B6 mice at 70% to 80% confluence. Western blots were performed on isolated juxtaglomerular cells using antibodies against either of the 2 calcium inhibitable isoforms of adenylyl cyclase, types-V and -VI. Only the antibody against adenylyl cyclase-V gave us a strong band at 120 kDa as expected. Immunolabeling in juxtaglomerular cells with confocal microscopy found immunofluorescence for the adenylyl cyclase-V–specific antibody compared with either negative controls or cells stained with the adenylyl cyclase-VI antibody. Reducing isolated juxtaglomerular intracellular calcium with 100 μmol/L of the cytosolic calcium chelator BAPTA-AM stimulated both cAMP (3.49±0.70 to 10.09±0.81 pmol/mL per milligram of protein; P<0.002) and renin release (1001.8±81.5 to 1648.0±139.1 ng of angiotensin I per milliliter per hour per milligram of protein; P<0.01). The selective adenylyl cyclase-V inhibitor NKY80 completely blocked both BAPTA-AM–stimulated cAMP formation and renin release. We conclude that lowering intracellular calcium is permissive, allowing an increased activity of the calcium-inhibitable isoform adenylyl cyclase-V (but not adenylyl cyclase-VI) in the juxtaglomerular cell, producing cAMP, which stimulates renin secretion.
Renin is the critical rate-limiting enzymatic factor in the formation of the potent vasoconstrictor angiotensin II. Secretion of renin from the juxtaglomerular (JG) cells is predominantly mediated by its stimulatory second messenger, cAMP,1 the product of adenylyl cyclase activity.2 However, unlike most secretory cell types, the renin release from the JG cell has a paradoxical interaction with intracellular calcium in that renin secretion is inhibited by increased intracellular calcium ([Ca+2]i) and conversely stimulated by decreased [Ca+2]i.3–5 The question of how [Ca+2]i and adenylyl cyclase might act or interact to regulate renin has been a long-standing subject of debate and investigation.1,5 Recently, using a preparation of primary cultures of isolated mouse JG cells that seem to completely maintain their phenotype and release renin,6 we found that a calcium-inhibitable form of adenylyl cyclase existed within the JG cell, suggesting that changes in [Ca+2]i regulate adenylyl cyclase activity, and this could account for this paradoxical relationship between [Ca+2]i and renin. We found that decreased [Ca+2]i leads to an increase in cAMP and, subsequently, renin release from isolated JG cells.
There are 9 isoforms of adenylyl cyclase. They all share a similar structure that includes an intracellular N terminus followed by 2 membrane-spanning domains alternating with 2 catalytic cytoplasmic loops.7 There are 2 adenylyl cyclase isoforms, type-V (AC-V) and type-VI (AC-VI), which are both inhibited by micromolar concentrations of [Ca+2]i.8 Both isoforms are found in lipid rafts in either the cell or intracellular membrane regions.9 In addition, AC-V and AC-VI share a high amino acid sequence identity, particularly in the regions that form the enzymes catalytic unit.10 However, the N termini of the 2 isoforms are quite divergent.7 It has been reported that, in nonexcitable cells, AC-VI colocalizes with the calcium entry channels in the cell membrane.11 Thus, whereas the 2 enzymes share many properties, they may also have different interactions within the cell and have different compartmentalization (such as in the surface or on intracellular membranes).
Previously,6 using an antibody that does not discriminate between the 2 isoforms, we found that either 1 or both of these calcium-inhibitable adenylyl cyclases existed within the JG cell, and their presence could explain the paradoxical effect of how decreasing [Ca+2]i could result in a cAMP-mediated stimulation of renin secretion. Although we observed a provocative colocalization of our antibody with renin on what appeared to be granule-like foci within the cytoplasm,6 we were unable to distinguish between the 2 isoforms to answer the critical question of which calcium-inhibitable isoform of adenylyl cyclase is in the JG cell and mediates this stimulation of renin secretion. Although either or both isoforms might exist within the JG cell, based on reports in some other cell types that AC-VI seems to localize in the cell membrane11 contrary to the cytoplasmic localization we observed,6 we hypothesized that it would be the AC-V isoform in the JG cells, and this isoform would be the enzymatic mediator of cAMP stimulation of renin secretion in response to decreased [Ca+2]i.
Isolation of Mouse JG Cells
Experiments were run using primary cultures of isolated mouse JG cells using our own modifications6 of techniques described previously.12,13 Eight- to 10-week-old C57/BL6 mice were obtained from Jackson Laboratories. C57/BL6 mice were given free access to food and water and were euthanized by cervical dislocation to avoid adverse effects of anesthesia on the harvested cells. Using a sterile technique, the kidneys were removed, and the renal cortex was dissected, minced, and then incubated in a digestion buffer6 containing 0.25% trypsin (activity: 15 500 U/mg; Sigma-Aldrich) and 0.1% collagenase (activity: 0.17 U/mg, type A; Roche) at 37°C for 75 minutes. The tissue was passed through 74-μm and then 22-μm sieves and then resuspended in 1-mL HEPES buffer.6 The JG cells were isolated using a 35% isoosmotic Percoll density gradient (Sigma). A band rich in JG cells was obtained at a density of 1.07 g/mL. The cells were washed of Percoll with HEPES buffer and resuspended in DMEM containing 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 5% FCS (Gibco-Invitrogen). The cells were divided equally into 250-μL aliquots and placed in 4 wells of a 24-well culture plate (Corning) to a density of 70% and incubated at 37°C in 5% CO2 for 48 hours. For experiments, the medium was replaced by 250 μL of fresh serum-free medium. All of the procedures were reviewed and approved by our institutional animal care and use committee and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Henry Ford Hospital’s animal facility is approved by the Association for Assessment and Accreditation of Laboratory Animal Care.
Renin Release From JG Cells
After 2-hour experimental incubations with serum-free medium containing 100 μmol/L of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine ([IBMX] Sigma), the media was removed, centrifuged, and then assayed for renin concentration using a radioimmunoassay for angiotensin I (Gamma Coat, DiaSorin) in the presence of excess of sheep angiotensinogen as described previously.14 Values for renin concentration (angiotensin I generated per milliliter of sample per hour of incubation) were corrected for JG cell total protein using a Coomassie Plus protein assay reagent kit (Pierce Biotechnology Inc).
cAMP Content in JG Cells
After the supernatant was removed, the cells were harvested by 2 consecutive 10-minute incubations in 100 μL of 0.1% HCl and then scraping the wells. JG cell lysate was centrifuged at 600g for 10 minutes, the supernatant collected, and cAMP content determined using an Endogen competitive ELISA (Pierce Biotechnology Inc). Values were correcting for JG cell total protein (as above).
AC-V and AC-VI Isoform Expression in JG Cells Using Western Blots
Isolated JG cells were suspended in lysis buffer (50 mmol/L Tris, pH 6.8/5% [vol/vol] glycerol/2% SDS) containing the protease inhibitors antipain (5 μg/mL), aprotinin (10 μg/mL), leupeptin (5 μg/mL), benzamidine (1 mmol/L), chymostatin (5 μg/mL), pepstatin-A (5 μg/mL), and 4-(2-aminoethyl)-benzenesulfonic acid hydrochloride (PF Block, 0.105 mmol/L) and incubated on ice for 10 minutes. Solubilized JG cell proteins (20 μg) were heated to 95°C for 5 minutes and the cell lysate subjected to polyacrylamide gel electrophoresis under reducing conditions. Proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (polyvinylidene fluoride, Invitrogen) overnight at 4°C. The membranes were blocked with 5% milk in tris-buffered saline tween for 1 hour at room temperature and then incubated with either an antibody selective for the AC-V isoform (Fabgennix International Inc)15 or an antibody selective for the AC-VI isoform (Abcam)16 in 1:1000 dilutions in 5% albumin overnight at 4°C. Primary antibodies were labeled with a horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:2000. The bands were detected using chemiluminescence and x-ray film. Each experiment was repeated twice.
Immunolabeling of AC-V and AC-VI in JG Cells
To determine whether the AC-V and/or AC-VI isoforms are in JG cells, we placed our primary cultures of JG cells for 48 hours on collagen IV–coated cover slips (Trevigen Inc). The medium was removed and the cells fixed for 30 minutes with freshly prepared 4% paraformaldehyde diluted in PBS then washed with tris-buffered saline tween 3 times for 5 minutes each. The fixed cells were permeabilized with 0.1% Triton X-100 for 20 minutes, then washed. Nonspecific binding was blocked with 5% normal goat serum for 30 minutes. The cells were washed and then incubated for 1 hour with either a primary antibody against the AC-V isoform (Fabgennix International Inc) or a primary antibody against the AC-VI isoform (Abcam) diluted 1:50 in 5% normal goat serum in tris-buffered saline tween. Cells were washed and incubated with a goat anti-rabbit antibody labeled with Alexa Fluor 568 red-orange fluorescent dye (Alexa Fluor, Invitrogen) diluted 1:100 in 5% BSA for 1 hour. After incubation with the secondary antibody, cells were washed, and the cover slips were mounted on slides with Fluoromount (Southern Biotech Associates, Inc). For controls, the primary antibodies were replaced by 5% goat serum. The preparations were examined by confocal microscopy (Visitech Confocal System) using excitation at 568 nm and emission measured at >590 nm. This imaging protocol was repeated 3 times.
Coimmunolabeling of Isoform AC-V and Renin
To test where in our cells the AC-V isoform localizes, immunofluorescence in JG cells using the AC-V antibody (as detailed above) and also an antibody against renin17 (Swant) was tested. The renin antibody was diluted 1:50 in 5% BSA and incubated with fixed JG cells and then incubated with a goat anti-mouse secondary antibody labeled with Alexa Fluor 488 green fluorescent dye (Alexa Fluor, Invitrogen) diluted 1:100 in 5% BSA in tris-buffered saline tween for 1 hour. Mounted samples were excited at 488 nm and emission measured at >500 nm to obtain images of the renin antibody and at 568 nm and emission measured at >590 nm for the AC-V antibody. The 2 images of a single cell were merged by overlaying tiff files in Photoshop (Adobe Systems) to show the relative intracellular localization of both the AC-V isoform and renin. Each experiment was repeated twice.
Inhibition of AC-V Activity
Isolated primary cultures of JG cells were incubated in serum-free medium containing 1 of the following 4 permutations: (1) media containing the phosphodiesterase inhibitor IBMX (100 μmol/L, control); (2) 100 μmol/L IBMX plus 100 μmol/L of the [Ca+2]i chelator BAPTA-AM (BAPTA)6,18; (3) 100 μmol/L IBMX plus 20 μmol/L of the selective AC-V isoform inhibitor 2-amino-7-(furanyl)-7,8-dihydro-5(6H)-quinazolinone (hereafter referred to as NKY80)19; and (4) 100 μmol/L of IBMX plus 100 μmol/L BAPTA plus 20 μmol/L NKY80. Cells were incubated for 2 hours, and then the media and cells were harvested for determination of intracellular cAMP, renin release, and total protein, as described above. The protocol was repeated 8 times.
Simple comparisons were determined using an unpaired Student t test. Multiple comparisons in the last protocol using BAPTA and NKY80 were run using ANOVA followed by a Bonferroni posthoc test. A P value (or adjusted P value) of <0.05 was considered statistically significant.
AC-V and AC-VI Isoform Expression in JG Cells Using Western Blots
Western blots using the selective antibody for the AC-V isoform (Figure 1A) identified a strong characteristic band at 120 kDa.15 Western blots using the selective antibody for the AC-VI isoform (Figure 1B) did not show a band at 130 kDa16 where it would be expected. A very faint band was barely apparent in this lane just below the 130-kDa site. These results support the predominance if not the exclusive presence of the type-V isoform in our preparation of isolated JG cells.
Immunolabeling of AC-V and AC-VI in JG Cells
Immunofluorescence and confocal microscopy were performed with the 2 selective antibodies for the AC-V and AC-VI isoforms. Figure 2 shows intense fluorescence in focal granule-like sites (Figure 2C) compared with minimal and diffuse staining of the secondary antibody control (Figure 2A). When immunofluorescence and confocal microscopy were performed using the antibody selective for the AC-VI isoform (Figure 3), the fluorescence was no different from negative controls using only the secondary antibody. The transmitted light image of each cell is provided beneath the fluorescent image. These data support the predominance of the type-V isoform in our preparation of isolated JG cells.
Coimmunolabeling of Isoform AC-V and Renin
Immunofluorescence and confocal microscopy with antibodies against the AC-V isoform and against renin are presented in Figure 4. A transmitted light image of the cell is presented in Figure 4A. Figure 4B shows the localization of renin in green, suggesting an anuclear distribution, which appears in granule-like foci throughout the cytoplasm, similar to what we have reported previously6 in isolated JG cells. Figure 4C shows the localization of the AC-V isoform, which also appears to be dispersed throughout the cytoplasm in granule-like foci, similar to the pattern seen in Figure 2. Figure 4D shows the 2 images merged, with yellow-to-orange staining representing colocalization. These data suggest a similar cytoplasmic localization for both renin and AC-V.
Inhibition of Adenylyl Cyclase V Activity
In Figure 5, the results of JG cell cAMP content (Figure 5, top) and renin release (Figure 5, bottom) are shown in response to a decrease in [Ca+2]i using the [Ca+2]i chelator BAPTA. Basal cAMP content in the isolated JG cells was 3.49±0.70 pmol/mL per milligram of protein, and cAMP was significantly increased 3-fold by BAPTA to 10.09±0.81 pmol/mL per milligram of protein (P<0.002). Basal renin release was 1001.8±81.5 ng of angiotensin I per milliliter per hour per milligram of protein, and renin release was significantly increased 65% by BAPTA to 1648.0±139.1 ng of angiotensin I per milliliter per hour per milligram of protein (P<0.01). These data are similar to what we have reported previously.6
Addition of the selective AC-V inhibitor NKY80 completely blocked the increase in both cAMP content and renin release from isolated JG cells (Figure 5). Basal cAMP content in the presence of NKY80 was 1.63±0.14 pmol/mL per milligram of protein (P<0.01 versus untreated controls). Renin release in the presence of NKY80 was 690.1±58.7 ng of angiotensin I per milliliter per hour per milligram of protein. Addition of BAPTA in the presence of NKY80 completely blocked the stimulation of JG cell cAMP content (2.84±0.26 pmol/mL per milligram of protein) and the increase in renin release (844.1±65.0 ng of angiotensin I per milliliter per hour per milligram of protein). These data are consistent with the concept that decreased [Ca+2]i stimulates activity of the calcium-inhibitable isoform AC-V, producing cAMP and stimulating renin release.
In this study, we hypothesized that the calcium-inhibitable isoform of adenylyl cyclase, AC-V, is present in JG cells, and this isoform would be the enzymatic mediator of cAMP stimulation of renin secretion in response to decreased [Ca+2]i. We found, using Western blots with selective antibodies for either the AC-V or AC-VI isoforms, that we could only clearly detect a band for AC-V, supporting the predominance if not the exclusive presence of the type-V isoform in our preparation of isolated JG cells. When immunofluorescence and confocal microscopy were performed using the same antibodies, we found a significant fluorescence for only the antibody against AC-V, supporting the predominance of the type-V isoform in JG cells. In addition, we found that this fluorescent image is dispersed throughout the cytoplasm in granule-like foci, similar to the pattern seen with an antibody against renin in the JG cell, suggesting a similar cytoplasmic localization for both renin and AC-V. Finally, stimulation of renin release from isolated JG cells using the [Ca+2]i chelator BAPTA resulted in significant increase in both cAMP and renin release, and both of these responses could be blocked with the selective AC-V inhibitor NKY80. All of these results combined are consistent with the hypothesis that decreased [Ca+2]i stimulates activity of the calcium-inhibitable isoform AC-V, producing cAMP and stimulating renin release.
There are 9 membrane-bound isoforms of adenylyl cyclase divided into 4 subfamilies. They all share a similar structure that includes an intracellular N terminus followed by 2 membrane-spanning domains (M1 and M2) alternating with 2 catalytic cytoplasmic loops (C1 and C2).7 Subfamily 3 includes the 2 isoforms, type-V and type-VI, which, in contrast to other groups, are both inhibited by micromolar concentrations of [Ca+2]i.8 In addition, AC-V and AC-VI share a high amino acid sequence identity, particularly in the regions that form the enzyme catalytic unit, and have similar properties beyond calcium’s inhibitory effect, such as being stimulated by Gαs and forskolin and inhibited by GαI-mediated processes.10 Both isoforms localize in lipid rafts in either the cell surface or intracellular membrane regions.9 However, the N termini of the 2 isoforms are quite divergent.7 Also, it has been suggested that protein kinase C enhances basal activity in AC-V but inhibits AC-VI activity in vivo.7 It has been reported that in nonexcitable cells AC-VI colocalizes with the calcium entry channels in the cell membrane.11 Thus, although the 2 enzymes share many properties, they may also have different interactions within the cell and have different compartmentalization (such as in the surface or on intracellular membranes). Although our previous data6 show calcium-inhibitable adenylyl cyclase in the cytoplasm of JG cells, we could not distinguish the prevalent isoform. In the present study, we proposed that it was type-V, primarily because of studies in other cell types, suggesting that type-VI was more likely to be found along the cell membrane functionally colocalized with Ca+2 entry channels of nonexcitable cells rather than in the cytoplasm.11 Individual cell types can simultaneously express multiple isoforms of adenylyl cyclase,20 and we do not suggest that AC-V is the exclusive isoform in the JG cell, although it seems to be the only 1 associated with decreased [Ca+2]i as a stimulus for renin release. Importantly, the calcium dependence of the AC-V isoform is not the only mechanism mediating the stimulation of renin release, because adenylyl cyclase can be stimulated by many other factors, and the level of [Ca+2]i may only modify its activity.
Although we do not see any immunolabeling of AC-VI in our JG cells, we do observe a very faint band in our Western blots near the expected 130-kDa site for AC-VI. This could mean that there is some small amount of AC-VI in these cells, that there is some contamination from non-JG cells that contain AC-VI from the cortex (our preparation is ≈85% pure6), or, because of the appearance of this band closer to the 120-kDa site for AC-V, there could be some cross-reactivity with the AC-V isoform.
Renal expression of AC-VI has been studied21 by RT-PCR in rat renal glomeruli and nephron segments isolated by microdissection, and its mRNA was present all along the nephron but more abundant in distal than in proximal segments. Chabardes et al21 reported that AC-VI is expressed in thick ascending limbs and collecting ducts. In contrast, the expression of renal type-V mRNA by RT-PCR was restricted to the glomerulus and to the initial portions of the collecting duct.21 None of these studies looked at the JG cells. Our previous study6 found that a calcium-inhibitable isoform(s) of adenylyl cyclase did exist within the JG cells, colocalized with renin in cytoplasmic granule-like foci, but we were unable to distinguish which isoform it was. Using antibodies to both renin and AC-V and AC-VI, we found similar results to our previous observations with the antibody against the type-V isoform but not with the type VI isoform. This anatomic localization of AC-V, which appears closely related to the distribution of renin, supports our functional data that it is involved in cellular mechanisms that regulate renin release from the JG cell.
In our previous work,6 we also found that decreasing [Ca+2]i stimulated both cAMP production and renin release in our preparation of JG cells, similar to what has been shown with only renin in other studies.5,18,22–24 Although we did not directly measure changes in [Ca+2]i, this technique of chelating [Ca+2]i is established18 and produces results similar to other techniques that lower [Ca+2]i. We find similar results in the present study, and, furthermore, we show that both cAMP generation and renin release induced by lowering [Ca+2]i can be blocked with a selective AC-V antagonist. Combined with the results from Western blots and the localization studies, we think these data provide a good case for the predominant effect of AC-V mediating the calcium paradox in regulating renin release from JG cells.
In conclusion, we hypothesized that that AC-V would be present in JG cells, and this isoform would be the enzymatic mediator of cAMP stimulation of renin secretion in response to decreased [Ca+2]i. We found that AC-V but not AC-VI is expressed in the cytoplasm, similar in localization to the distribution of renin in isolated JG cells, and that stimulation of both JG cell cAMP production and renin release by decreasing [Ca+2]i could be blocked by a selective AC-V inhibitor. These data are all consistent with AC-V being the adenylyl cyclase isoform responsible for the interaction among changing [Ca+2]i, cAMP production, and renin secretion.
The basis of the paradoxical inverse relationship between the concentration of [Ca+2]i and renin secretion has been a perplexing question for decades.1 The present study not only confirms that this relationship is because of the activity of a calcium-inhibitable isoform of adenylyl cyclase but finally identifies the specific isoform, type V. Hopefully, understanding this process along with the characteristics and interactions unique to AC-V will help us further unravel the mechanisms by which increased cAMP formation in the JG cell actually lead to regulating renin secretion. Understanding the mechanisms controlling renin is critical to our search for integrating cardiovascular and renal function in the control of blood pressure and cardiorenal homeostasis.
Source of Funding
This research was supported by National Institutes of Health grant RO-1 HL076469.
- Received October 16, 2006.
- Revision received November 9, 2006.
- Accepted December 4, 2006.
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