Decreased Intracellular Calcium Stimulates Renin Release via Calcium-Inhibitable Adenylyl Cyclase
Intracellular calcium and cAMP are the 2 second messengers that regulate renin release; cAMP stimulates renin release from juxtaglomerular (JG) cells, whereas increased intracellular calcium inhibits it. We hypothesized that decreased intracellular calcium acts by activating calcium-inhibitable isoforms of adenylyl cyclase, increasing cAMP, and stimulating renin secretion. We used a primary culture of JG cells isolated from C-57/B6 mice. Cells were plated to a density of 70% in serum-free medium and incubated for 2 hours with or without 100 μmol/L of the cytosolic calcium chelator 5′5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Intracellular cAMP content was 4.04±0.92 pM/mL per milligram of protein, and it increased by125±33% (P<0.01) with BAPTA-AM. Basal renin was 1.28±0.40 μg of angiotensin I per milliliter per hour per milligram of protein, and BAPTA-AM increased it by 182±62% (P<0.025). Western blots using an antibody that recognizes adenylyl cyclase types V and VI yielded a characteristic band of ≈135 kDa. When primary cultures of isolated JG cells were tested for the calcium-inhibitable isoforms of adenylyl cyclase, they showed intense focal cytoplasmic staining. Cells stained for both renin and adenylyl cyclase V/VI showed colocalization in the cytoplasm, primarily on the granules. An adenylyl cyclase inhibitor (SQ 22,536) completely blocked BAPTA-AM–stimulated renin release and JG cell cAMP content. We conclude that calcium-inhibitable isoform(s) of adenylyl cyclase (types V and/or VI) exist within the JG cell. Thus, decreased intracellular calcium stimulates adenylyl cyclase, resulting in cAMP synthesis and, consequently, renin release.
Renin is the rate-limiting enzymatic step in the formation of angiotensin (Ang); thus, control of renin secretion by the kidney is a critical element in regulating systemic blood pressure and renal function. The common element in all of the renin-stimulating pathways is the second messenger cAMP,1 the product of adenylyl cyclase activity.2 However, it is also well established that renin secretion by the JG cells, unlike almost all secretory cells, is inversely related to intracellular calcium concentration such that paradoxically elevated intracellular calcium is a potent inhibitor of renin release.3–5 Increased calcium in the juxtaglomerular (JG) cells suppresses basal renin release and blunts stimulation of renin secretion.6–9 Decreased JG cell intracellular calcium increases basal renin secretion and amplifies stimulated renin levels.3,6,9–11 Although the influence of these 2 “second messenger” regulatory signals, cAMP and calcium, has been established for years,1,5,6,10,12 the precise nature of their interactions is unresolved and an area of considerable interest and debate.
There are at least 9 isoforms of adenylyl cyclase,2 including 2 (types V and VI) that are inhibited by increased intracellular calcium.13 Because renin release is inhibited by increased intracellular calcium and stimulated by cAMP, we hypothesized that reducing intracellular calcium stimulates renin release by activating a calcium-inhibitable adenylyl cyclase, types V and/or VI in JG cells, enhancing cAMP levels, and thereby stimulating renin release. To test this hypothesis, we used primary cultures of isolated mouse JG cells, which exhibit the classic phenotypic character of the JG cell but are unencumbered by the many extracellular signaling pathways that can influence renin secretion in vivo or in less homogeneous in vitro preparations. Our results provide a unique answer to this longstanding question of how these 2 classic second messengers interact to control the release of renin from the JG cell.
Isolation of Mouse JG Cells
Eight- to 10-week-old C57/BL6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Primary culture of mouse isolated JG cells was used, following a protocol modified from methods described previously.14,15 Typically, 4 C57/BL6 mice given free access to food and water were euthanized by cervical dislocation to avoid adverse effects of anesthesia on the harvested cells. Using a sterile technique, the kidneys were removed, decapsulated, and the renal cortex dissected. Cortical tissue was minced and then incubated with gentle stirring in a digestion buffer (130 mmol/L of NaCl, 5 mmol/L of KCl, 2 mmol/L of CaCl2, 10 mmol/L of glucose, 20 mmol/L of sucrose, 10 mmol/L of HEPES [pH 7.4], and osmolality of 310 milliosmol) containing 0.25% trypsin (activity: 15 500 U/mg, Sigma-Aldrich) and 0.1% collagenase (activity: 0.17 U/mg, type A; Roche Applied Science) at 37°C for 75 minutes. The digestion buffer was replenished every 15 minutes. After enzymatic dissociation, the tissue was passed first through a 74-μm and then through a 22-μm nylon mesh sieve. The retrieved cells were washed and resuspended in 1 mL of HEPES buffer containing 130 mmol/L of NaCl, 5 mmol/L of KCl, 10 mmol/L of glucose, 20 mmol/L of sucrose, and 10 mmol/L of Hepes (pH 7.4), with osmolality 310 milliosmol. The cell suspension was separated using density centrifugation with 25 mL of a 35% isoosmotic Percoll density gradient (Sigma). A companion volume-matched tube with Percoll marker beads was also run. After 25 minutes and ultracentrifugation at 4°C and 17 000g using a 50.2Ti rotor (Beckman Coulter), a cellular layer rich in JG cells was obtained at a density of 1.07 g/mL. After Percoll was washed from the cells with Hepes buffer, they were 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%. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. After 48 hours, the culture medium was removed and 250-μL fresh prewarmed culture medium without FCS was added. Experiments were performed on primary cultures of mouse isolated JG cells in serum-free medium. These procedures were reviewed by our institutional animal care and use committee and adhere to the guiding principles in the care and use of experimental animals. Henry Ford Hospital animal facility is Association for Assessment and Accreditation of Laboratory Care (AAALAC) approved.
Renin Release From Isolated Mouse JG Cells
JG cells were incubated for 2 hours with serum-free medium (with or without drugs), containing 100 μmol/L of 3-isobutyl-1-methyl-xanthine ([IBMX] Sigma) to prevent phosphodiesterase degradation of cAMP. After incubation, the supernatant was centrifuged to remove cellular debris and assayed for renin using an adaptation of an assay previously reported by us16 in which the sample is incubated with a excess of sheep angiotensinogen and then tested for generation of Ang I using a Gamma Coat radioimmunoassay kit (DiaSorin). Values for renin concentration (Ang I generated per milliliter of sample per hour of incubation) were normalized by correcting for JG cell total protein.
cAMP Content in Isolated Mouse JG Cells
cAMP content of isolated JG cells was determined in the presence of 100 μmol/L of IBMX to prevent cAMP degradation by phosphodiesterases. Once the supernatant was removed for renin determination, the cells were harvested by 2 consecutive 10-minute incubations in 100 μL of 0.1% HCl and scraping the wells. The lysate was centrifuged at 600g for 10 minutes, the supernatant collected, and cAMP content determined using a colorimetric immunoassay kit (R&D Systems). Values were correcting for JG cell total protein.
All of the values were normalized by JG cell protein concentration. Protein concentration in JG cellular lysates was determined using a Coomassie Plus protein assay reagent kit (Pierce Biotechnology Inc).
Characterization of Isolated JG Cells
Transmission Electron Microscopy
Isolated JG cells from primary cultures were fixed with 4% paraformaldehyde then postfixed in 2% osmium tetroxide, dehydrated through a gradient of ethanol, and embedded in araldite after the methods of Tickoo et al.17 Ultrathin sections were obtained with an LKB Nova ultra microtome, stained with lead citrate and uranyl acetate, and viewed with a Philips 208 electron microscope by our institutional electron microscopic imaging core in the Department of Pathology. Images were taken at ×13 500 magnification.
Immunolabeling of Cultured JG Cells
To demonstrate the presence of renin in our preparation, JG cells were cultured for 48 hours on collagen IV–coated coverslips (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 with Tween-20 (TBST) 3 times for 5 minutes each. The fixed cells were permeabilized with PBS containing 0.1% Triton X-100 for 20 minutes, then washed. Nonspecific binding was blocked with TBST and 5% BSA for 30 minutes. The cells were washed and then incubated for 1 hour with a primary antibody against mouse renin18 (Swant, Bellinzona, Switzerland), diluted 1:50 in 5% BSA in TBST. Cells were washed and 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 TBST for 1 hour. After incubation with the secondary antibody, cells were washed, and the coverslips were mounted on slides with Fluoromount (Southern Biotech Associates, Inc). The following controls were run to test the specificity of the binding: primary and secondary antibodies were replaced by TBST 5% BSA, and the primary antibodies were replaced by TBST 5% BSA. The preparations were examined by confocal microscopy (Visitech Confocal System). Samples were excited at 488 nm and emission measured at >500 nm. This imaging protocol was repeated for 6 preparations.
Stimulated Adenylyl Cyclase and Renin Release
We exposed primary cultures of isolated JG cells to serum-free medium plus 100 μmol/L of IBMX (control, n=6) or 100 μmol/L IBMX plus 1 μmol/L 7β-acetoxy-8,13-epoxy-1α,6β,9α-trihydroxy-labd-14-ene-11-1 (forskolin, Sigma) to directly stimulate adenylyl cyclase (n=6) for 2 hours. Additional experiments were run using 100 μmol/L of IBMX plus 400 μmol/L of the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, n=6)19 or 100 μmol/L of IBMX and 1 μmol/L of forskolin plus 400 μmol/L of SQ 22 536 (n=6). Renin release into the medium was determined as described above. Cells were harvested for determination of cAMP content (as described above) and for total protein determination.
Chelating Intracellular Calcium
We placed primary cultures of isolated JG cells in serum-free medium plus 100 μmol/L of IBMX (control, n=7) or 100 μmol/L of IBMX plus 100 μmol/L of the intracellular calcium chelator 5, 5′-dimethyl 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA-AM)20 (Invitrogen) for 2 hours. Cellular cAMP content, renin release into the medium, and total protein were determined as described above.
Western Blot of Adenylyl Cyclase Isoforms
Isolated JG cells were resuspended in lysis buffer (50 mmol/L of Tris [pH 6.8]/5% [vol/vol] glycerol/2% SDS containing the following protease inhibitors: 5 μg/mL of antipain, 10 μg/mL of aprotinin, 5 μg/mL of leupeptin, 1 mmol/L of benzamidine, 5 μg/mL of chymostatin, 5 μg/mL of pepstatin-A, and 0.105 mmol/L of 4-(2-aminoethyl)-benzenesulfonic acid hydrochloride [PF Block]) and incubated for 10 minutes on ice. The samples were centrifuged at 13 000g for 5 minutes at 4°C. 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 TBST for 1 hour at room temperature and then incubated with the adenylyl cyclase V/VI antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000 dilutions in 5% albumin) overnight at 4°C. Primary antibodies were labeled with an 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. These experiments were repeated 3 times. Additional experiments were run using positive controls for the adenylyl cyclase V/VI antibody. Twenty microliters of mouse brain extract in SDS-PAGE (Santa Cruz) and 20 μL of mouse heart extract in SDS-PAGE (Santa Cruz) were used as positive controls. Extracts were heated to 95°C for 5 minutes and subjected to polyacrylamide gel electrophoresis under reducing conditions. Proteins were electrophoretically transferred (overnight at 4°C) to a polyvinylidene difluoride membrane (polyvinylidene fluoride, Invitrogen). The membranes were blocked for 1 hour at room temperature and were then incubated with an antibody to adenyl cyclase V/VI (Santa Cruz, 1:1000 dilution in 5% albumin) overnight at 4°C. Detection was achieved using a horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:2000. The bands were identified by chemiluminescence and exposed to x-ray film.
Immunolabeling of Adenylyl Cyclase V and/or VI in JG Cells
Immunolabeling of calcium-inhibitable isoforms of adenylyl cyclase in isolated JG cells was performed using a protocol similar to the renin immunolabeling (see above) with the following modifications. Specific binding sites were blocked with 5% normal goat serum in TBST. The primary antibody13 from Santa Cruz Biotechnology was specific for both calcium-inhibitable isoforms of anti-adenylyl cyclase, types V and VI, and was used at a dilution of 1:50 in TBST 5% normal goat serum. The secondary antibody was goat anti-rabbit labeled with Alexa Fluor 568 red-orange fluorescent dye (Alexa Fluor, Invitrogen) with excitation at 568 nm and emission measured at >590 nm.
Inhibition of Adenylyl Cyclase
After 48-hour incubation, cells were transferred to serum-free medium containing 1 of the following 4 permutations: (1) 100 μmol/L of IBMX (control); (2) 100 μmol/L of IBMX plus 100 μmol/L of BAPTA-AM; (3) 100 μmol/L of IBMX plus 400 μmol/L of the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536) 19; or (4) 100 μmol/L of IBMX and 100 μmol/L of BAPTA-AM plus 400 μmol/L of SQ 22,536. Cells were incubated for 2 hours and then harvested for determination of intracellular cAMP, renin release, and total protein as described above.
Statistical analysis of the changes in both JG cell renin release and cAMP concentration was measured by Student’s paired t test to evaluate values between matched cultures with and without treatment. A P value <0.05 was considered significant.
Characterization of Renin-Containing JG Cells
We tested a number of bands in the 35% Percoll gradient for renin content and found renin activity in 3; at a density of 1.04 g/mL, we detected renin content of 0.54±0.18 μg of Ang I per milliliter per hour per milligram of protein. Similarly, at a density of 1.055 g/mL we detected renin content of 0.86±0.23 μg of Ang I per milliliter per hour per milligram protein. Finally, at a density of 1.07 g/mL we detected renin content of 15.00±1.00 μg of Ang I milliliter per hour per milligram, an amount at least 18-fold greater than in any other band. These results were highly reproducible over 6 repetitions of the isolation protocol. Thus, for all of the subsequent studies we harvested cells from the 1.07 g/mL density band.
Cells with high renin content obtained from the 1.07 g/mL band were visualized by transmission electron microscopy (Figure 1A). They had a characteristic appearance of JG cells in culture, including cytoplasm filled with dense granules (Figure 1B). When they were characterized with an antibody specific for renin (Figure 1C), they showed fluorescence that was primarily compacted into granule-like foci within the cytoplasm. By counting the number of cells in our primary culture obtained from the 1.07-g/mL density band stained positive for renin, we determined that the purity of the preparation was ≈82.9±3.5%, suggesting that whereas it was not pure JG cells, it was highly enriched with these cells.
Because cAMP is a second messenger that mediates stimulation of renin release, the JG cells should release renin in response to stimulation by adenylyl cyclase. We exposed primary cultures of JG cells to 1 μmol/L of forskolin for 2 hours, comparing them with untreated time control cultures. Initial studies in the absence of phosphodiesterase inhibition yielded marginal results. However, by adding 100 μmol/L of the phosphodiesterase inhibitor IBMX to all of the incubations increased basal renin release to 553.9±134.3 ng of Ang I per milliliter per hour per milligram of protein. As shown in Figure 2, adding forskolin nearly doubled renin release (P<0.025) to 932.6±156.2 ng Ang I per milliliter per hour per milligram of protein. Basal cAMP measurements in the presence of IBMX were 4.63±1.30 pM/mL per milligram protein, and addition of forskolin stimulated an 11-fold increase in cAMP (Figure 2) to 44.46±12.94 pM/mL per milligram of protein (P<0.005). Thus, all of the subsequent studies were carried out in the presence of IBMX.
Additional experiments were carried out in the same preparations with the addition of 400 μmol of the adenylyl cyclase inhibitor SQ 22,536.19 Basal renin release in the presence of the inhibitor was 331.1±82.2 ng of Ang I per milliliter per hour per milligram of protein (Figure 2), and the addition of forskolin in the presence of the inhibitor blocked the stimulation of renin (509.1±48.9 ng of Ang I per milliliter per hour per milligram of protein) by forskolin. Basal cAMP content was reduced in the presence of the adenylyl cyclase inhibitor (2.35±0.68 pM/mL per milligram of protein), and addition of forskolin (Figure 2) generated only a marginal increase in cAMP back to basal levels (4.71±1.59 pM/mL per milligram of protein). Thus, inhibition of adenylyl cyclase activity in the JG cells blocked both generation of cAMP and the release of renin.
Intracellular Calcium and Adenylyl Cyclase
Intracellular calcium concentration of primary cultures of isolated mouse JG cells was reduced by adding 100 μmol/L of the intracellular calcium chelator, 5′5 dimethyl BAPTA-AM.20 JG cell intracellular cAMP content in untreated controls was 4.04±0.92 pmol/mL per milligram of protein and was increased by 125±33% to 9.21±3.39 pmol/mL per milligram of protein (P<0.01) by BAPTA treatment (Figure 3). Thus, decreasing JG intracellular calcium led to stimulation of adenylyl cyclase as indicated by the increase in cAMP. Basal renin release in controls was 1.28±0.40 μg of Ang I per milliliter per hour per milligram of protein. Addition of BAPTA-AM increased renin release by 182±62% to 2.57±0.65 μg of Ang I per milliliter per hour per milligram of protein (P<0.025; Figure 3). Thus, the elevation in JG cell cAMP was accompanied by increased renin release.
Because we found that reduced intracellular calcium stimulated adenylyl cyclase activity, we looked for evidence of calcium-inhibitable isoforms in the JG cells. Western blots were performed using an adenylyl cyclase V/VI antibody from Santa Cruz.13 Figure 4A identifies a characteristic band at 135 kDa, showing the presence of either/or both types V and VI.12,21 Each Western blot gave similar positive results. Additional positive controls for types V and VI using mouse heart and brain standards were also positive (Figure 4B). Although this antibody does not discern which specific calcium-inhibitable isoform is present, it clearly indicates that at least 1 such isoform is present in the JG cells.
Because Western blots supplied evidence for the presence of one or both calcium-inhibitable isoform(s), we used immunolabeling and confocal microscopy to image the isoforms in the cells. JG cells grown on coated coverslips and stained for immunofluorescence are shown in Figure 5. Three separate experiments showed intense focal cytoplasmic staining for adenylyl cyclase V/VI in the JG cells.
In other studies, isolated JG cells were grown on coverslips and stained for both renin and adenylyl cyclase V/VI. Figure 6 shows the same cell stained for adenylyl cyclase V/VI (red) and renin (green), along with a combined image illustrating colocalization of renin and adenylyl cyclase V/VI (shown in yellow). This figure suggests that calcium-inhibitable adenylyl cyclase colocalizes with renin and seems to be associated with the granules within the cytoplasm.
Although we found that decreased intracellular calcium stimulates adenylyl cyclase, we wanted to rule out any effect of decreased intracellular calcium independent of cAMP. For this, we examined renin and cAMP responses to decreased intracellular calcium in the presence of an adenylyl cyclase inhibitor. cAMP (Figure 7, top) and renin (Figure 7, bottom) were stimulated by reducing intracellular calcium using 100 μmol/L of BAPTA-AM. Basal JG cell cAMP content was 4.33±0.54 pM/mL per milligram of protein, and BAPTA-AM increased it by 40.4±5.6% to 6.13±0.75 pM/mL per milligram of protein (P<0.005). Basal renin release was 159.1±20.6 ng of Ang I per milliliter per hour per milligram of protein, and BAPTA-AM increased it by 108.6±34.4% to 304.7±42.8 ng of Ang I per milliliter per hour per milligram of protein (P<0.025).
Addition of 400 μmol of the adenylyl cyclase inhibitor SQ 22,53619 completely blocked the increase in JG cell cAMP content (Figure 7). JG cell cAMP content in the presence of SQ 22,536 was reduced by 28% from basal to 3.11±0.05 pmol/mL per milligram of protein and showed no further change when BAPTA-AM was added (3.08±0.43 pmol/mL per milligram of protein). Renin release was 141.9±31.6 ng of Ang I per milliliter per hour per milligram of protein in the presence of SQ 22,536 and remained unchanged with BAPTA-AM (168.2±54.7 ng of Ang I per milliliter per hour per milligram of protein). Thus, inhibiting adenylyl cyclase completely eliminated the renin response to decreased JG cell intracellular calcium.
We first established and characterized a method of primary culture of mouse isolated JG cells in our laboratory based on the original techniques described previously.14,15 To improve recovery and reliability, we modified the buffers, stabilizing the digestion step and using marker beads to more clearly identify the specific band rich in JG cells within the Percoll gradient. We were able to identify a band of cells that contained at least 18 times more renin than other bands. This more than doubled the amount of tissue harvested, improving the yield from 30% JG cells in the primary culture to >80% and also greatly improving adherence of the cells to the culture surface. They had a characteristic JG-like appearance,22 with dense granules filling the cytoplasm. An antibody against renin stained the cytoplasm of permeabilized JG cells in a pattern similar to the renin-containing granules. The JG cells contain cAMP and released renin, and stimulating adenylyl cyclase with forskolin significantly increased both cAMP and renin release. These responses were both blocked with the adenylyl cyclase inhibitor SQ 22,536.19 Thus, our primary culture of mouse JG cells behaves as would be expected.
Preliminary studies showed that decreasing extracellular calcium to a nominal 0 concentration in the medium7,11 doubled basal renin release from primary cultures of JG cells (data not shown), whereas increasing calcium in the medium resulted in JG cells lifting off from the surface making the preparation useless. To change intracellular calcium without stressing the cells by changing the bathing medium, we used the technique of Moe et al20 to chelate intracellular calcium and found that not only was renin release significantly increased, but so was cAMP content. This provided strong evidence that the interaction between the 2 second messenger pathways, intracellular calcium and cAMP, is mediated first by decreased intracellular calcium, which could increase adenylyl cyclase activity, cAMP formation, and, consequently, renin release. This relationship of intracellular calcium as a modulator of adenylyl cyclase activity is consistent with a number of previous studies examining the interaction of these 2 second messengers.3–12 However, our observation is unique, showing that the decrease in intracellular calcium actually stimulates adenylyl cyclase activity to increase JG cell cAMP content.
The logical explanation for stimulation of JG cell cAMP content by decreased intracellular calcium seemed to be the presence of calcium-inhibitable adenylyl cyclase isoforms (types V and VI). These isoforms have been characterized as inhibited by Gi, stimulated by forskolin, GTP analogues, and other hormones or factors, which are typically adenylyl cyclase agonists.21,23,24 They respond to submicromolar changes in intracellular calcium,24 consistent with the small changes calculated by Park et al,25 which can alter renin secretion.
We performed Western blots of JG cell protein using an established antibody that identified either or both adenylyl cyclase isoforms13 and obtained a strong band in the region of 130 to 139 kDa molecular weight, consistent with the 2 calcium-inhibitable isoforms.13 Combining the same antibody with confocal microscopy and immunofluorescence, we obtained a strong positive focal signal in the cytoplasm of the JG cells. Coupled with the Western blots, we believe our findings present novel and compelling evidence that JG cells contain significant amounts of calcium-inhibitable adenylyl cyclase in the cytoplasm, consistent with our hypothesis. Determining whether 1 or both isoforms are involved will take considerably more study, which is beyond the scope of the current work. Nevertheless, our data provide a novel explanation for the calcium paradox of the JG cell.
Using antibodies to both renin and calcium-inhibitable adenylyl cyclase, we examined their colocalization within the JG cell. We expected to find that adenylyl cyclase was localized to the cell membrane,26 and were surprised to find that it colocalized with renin, primarily on the renin-containing granules. It seems apparent from this anatomic colocalization that the calcium-inhibitable isoform(s) are closely related to cellular mechanisms that regulate renin in the JG cell.
We found that nonselective inhibition of adenylyl cyclase activity19 blocked both the forskolin stimulation of adenylyl cyclase activity and renin release and the effect of decreasing intracellular calcium on both increasing cAMP and stimulating renin release. Churchill, in his seminal review,1 proposed several theoretical pathways by which cAMP and intracellular calcium could interact. Our data with the adenylyl cyclase inhibitor suggest that there is no alternative (non-cAMP–mediated) pathway by which a decrease in intracellular calcium might stimulate renin release. Again, this observation supports our hypothesis that the decrease in calcium activates adenylyl cyclase, resulting in cAMP formation and, subsequently, renin release. It suggests that basal levels of intracellular calcium quench adenylyl cyclase activity, as predicted by Park et al,25 and that reducing the intracellular calcium further stimulates (or disinhibits) its enzymatic activity. The actual mechanism by which cAMP stimulates the release of renin is not understood beyond its effect on cAMP-dependent protein kinase A or interacting with some other cAMP-binding protein promoting an undefined cascade that results in renin release from storage granules in the JG cells. As a note of caution, we do not suggest that this pathway via calcium-inhibitable adenylyl cyclase is the sole mechanism mediating the stimulation of renin release. Certainly adenylyl cyclase can be stimulated by many other factors for which the level of intracellular calcium may only serve as permissive. In addition, the JG cell surely contains other isoforms of adenylyl cyclase,2 which may not be associated with this pathway mediating renin release. Our studies only address the specific regulation of adenylyl cyclase activity associated with changing intracellular calcium, and these observations should ultimately fit in to a broader scheme, which defines all of the integrated pathways regulating renin.
In conclusion, we hypothesized that calcium-inhibitable isoform(s) of adenylyl cyclase (types V and/or VI) exist within the JG cell. Decreased intracellular calcium leads to a stimulation of adenylyl cyclase, increasing cAMP synthesis and, consequently, renin release. We show that decreased intracellular calcium increases adenylyl cyclase activity, cAMP synthesis, and release of renin from the JG cell. Western blots and immunofluorescence showed that calcium-inhibitable isoforms of adenylyl cyclase are present in the cytoplasm of the JG cell and, furthermore, that they seem to colocalize with renin in association with the renin-containing granules. Finally, inhibition of adenylyl cyclase completely blocks the effect of decreased intracellular calcium on renin, suggesting that it is the interaction of intracellular calcium and adenylyl cyclase type V and/or VI, rather than some undefined cAMP-independent effect that mediates this paradoxical ability of decreased intracellular calcium to stimulate renin release from the JG cell.
In most secretory cells, calcium is an important mediator of secretion. The calcium paradox, or the inverse relationship between secretion and intracellular calcium, is present in only 2 of the hundreds of secretory cell types: the parathyroid and the renal JG cell.2 The explanation for this unusual cellular phenomenon has escaped resolution for decades and has been the topic of hundreds of studies and numerous reviews. Our study provides evidence for an exquisitely obvious explanation, that selective calcium-inhibitable isoforms of adenylyl cyclase exist in the JG cell and mediate the effect of intracellular calcium on renin secretion. It is quite possible that the same explanation also holds for the parathyroid. In any event, we believe our results resolve one of the great long-standing conundrums of renin regulation and provide a logical resolution to this problem.
Sources of Funding
M.C.O.C. and W.H.B. are recipients of National Institutes of Health research grant RO-1 HL076469. J.L.G. is a principal investigator on National Institutes of Health grant RO-1, HL070985 and project investigator in National Institutes of Health program project HL28982.
- Received May 27, 2006.
- Revision received June 12, 2006.
- Accepted October 12, 2006.
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