Endothelin inhibits renin release from juxtaglomerular cells via endothelin receptors A and B via a transient receptor potential canonical-mediated pathway.

Renin is the rate-limiting step in the production of angiotensin II: a critical element in the regulation of blood pressure and in the pathogenesis of hypertension. Renin release from the juxtaglomerular (JG) cell is stimulated by the second messenger cAMP and inhibited by increases in calcium (Ca). Endothelins (ETs) inhibit renin release in a Ca-dependent manner. JG cells contain multiple isoforms of canonical transient receptor potential (TRPC) Ca-permeable channels. The proposed hypothesis is that endothelin inhibits renin release by activating TRPC store-operated Ca channels. RT-PCR and immunofluorescence revealed expression of both ETA and ETB receptors in mouse JG cells. Incubation of primary cultures of JG cells with ET-1 (10 nmol/L) decreased renin release by 28%. Addition of either an ETA or an ETB receptor blocker completely prevented the ET inhibition of renin release. Incubation with the TRPC blocker (SKF 96365, 50 µmol/L) completely reversed the Ca-mediated inhibition of renin release by ETs. These results suggest that endothelin inhibits renin release from JG cells via both ETA and ETB receptors, which leads to the activation of TRPC store-operated Ca channels.

Recruited renin-containing renal microvascular cells demonstrate the calcium paradox regulatory phenotype.

Renin is the critical regulatory enzyme for production of angiotensin (Ang)-II, a potent vasoconstrictor involved in regulating blood pressure and in the pathogenesis of hypertension. Chronic sodium deprivation enhances renin secretion from the kidney, due to recruitment of additional cells from the afferent renal microvasculature to become renin-producing rather than just increasing release from existing juxtaglomerular (JG) cells. JG cells secrete renin inversely proportional to extra- and intracellular calcium, a unique phenomenon characteristic of the JG regulatory phenotype known as the "calcium paradox." It is not known if renin secreted from recruited renin-containing cells is regulated similarly to native JG cells, and therefore acquires this JG cell phenotype. We hypothesized that non-JG cells in renal microvessels recruited to produce renin in response to chronic dietary sodium restriction would demonstrate the calcium paradox, characteristic of the JG cell phenotype. Histology showed recruitment of upstream arteriolar renin in response to sodium restriction compared to normal-diet rats. Renin fluorescence intensity increased 53% in cortices of sodium-restricted rats (P<0.001). We measured renin release from rat afferent microvessels, isolated using iron oxide nanopowder and incubated in either normal or low-calcium media. Basal renin release from normal sodium-diet rat microvessels in normal calcium media was 298.1±44.6 ng AngI/mL/hour/mg protein, and in low-calcium media increased 39% to 415.9±71.4 ng AngI/mL/hour/mg protein (P<0.025). Renin released from sodium-restricted rat microvessels increased 50% compared to samples from normal-diet rats (P<0.04). Renin release in normal calcium media was 447.0±54.3 ng AngI/mL/hour/mg protein, and in low-calcium media increased 36% to 607.6±96.1 ng AngI/mL/hour/mg protein (P<0.05). Thus, renin-containing cells recruited in the afferent microvasculature not only express and secrete renin but demonstrate the calcium paradox, suggesting renin secretion from recruited renin-containing cells share the JG phenotype for regulating renin secretion.

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

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 A2AR and A2BR. 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.

Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP(3) pathway and the ryanodine receptor.

The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P < 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P < 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 ± 8 to 118 ± 22% of control (P < 0.01). Combining inhibition of IP(3) and RyR was not additive. G(i) inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 ± 12 to 41 ± 8% of control, P < 0.001). We conclude stimulating JG cell CaSR activates G(q), initiating the PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

Acute activation of the calcium-sensing receptor inhibits plasma renin activity in vivo.

In vitro, the renin-secreting juxtaglomerular cells express the calcium-sensing receptor, and its activation with the calcimimetic cinacalcet inhibits renin release. To test whether the activation of calcium-sensing receptor similarly inhibits plasma renin activity (PRA) in vivo, we hypothesized that the calcium-sensing receptor is expressed in juxtaglomerular cells in vivo, and acutely administered cinacalcet would inhibit renin activity in anesthetized rats. Since cinacalcet inhibits parathyroid hormone, which may stimulate renin activity, we sought to determine whether cinacalcet inhibits renin activity by decreasing parathyroid hormone. Lastly, we hypothesized that chronically administered cinacalcet would inhibit basal and stimulated renin in conscious rats. Calcium-sensing receptors and renin were localized in the same juxtaglomerular cells using immunofluorescence in rat cortical slices fixed in vivo. Cinacalcet was administered acutely via intravenous bolus in anesthetized rats and chronically in conscious rats by oral gavage. Acute administration of cinacalcet decreased basal renin activity from 13.6 ± 2.4 to 6.1 ± 1.1 ng ANG I·ml(-1)·h(-1) (P < 0.001). Likewise, cinacalcet decreased furosemide-stimulated renin from 30.6 ± 2.3 to 21.3 ± 2.3 ng ANG I·ml(-1)·h(-1) (P < 0.001). In parathyroidectomized rats, cinacalcet decreased renin activity from 9.3 ± 1.3 to 5.2 ± 0.5 ng ANG I·ml(-1)·h(-1) (P < 0.05) similar to sham-operated controls (13.5 ± 2.2 to 6.6 ± 0.8 ng ANG I·ml(-1)·h(-1), P < 0.05). Chronic administration of cinacalcet over 7 days had no significant effect on PRA under basal or stimulated conditions. In conclusion, calcium-sensing receptors are expressed in juxtaglomerular cells in vivo, and acute activation of these receptors with cinacalcet inhibits PRA in anesthetized rats, independent of parathyroid hormone.

Calcium-dependent phosphodiesterase 1C inhibits renin release from isolated juxtaglomerular cells.

Renin release from the juxtaglomerular (JG) cell is stimulated by the second messenger cAMP and inhibited by calcium. We previously showed JG cells contain a calcium sensing receptor (CaSR), which, when stimulated, decreases cAMP formation and inhibits renin release. We hypothesize CaSR activation decreases cAMP and renin release, in part, by stimulating a calcium calmodulin-activated phosphodiesterase 1 (PDE1). We incubated our primary culture of JG cells with two selective PDE1 inhibitors [8-methoxymethil-IBMX (8-MM-IBMX; 20 microM) and vinpocetine (40 microM)] and the calmodulin inhibitor W-7 (10 microM) and measured cAMP and renin release. Stimulation of the JG cell CaSR with the calcimimetic cinacalcet (1 microM) resulted in decreased cAMP from a basal of 1.13 +/- 0.14 to 0.69 +/- 0.08 pM/mg protein (P < 0.001) and in renin release from 0.89 +/- 0.16 to 0.38 +/- 0.08 microg ANG I/mlxh(-1)xmg protein(-1) (P < 0.001). However, the addition of 8-MM-IBMX with cinacalcet returned both cAMP (1.10 +/- 0.19 pM/mg protein) and renin (0.57 +/- 0.16 microg ANG I/mlxh(-1)xmg protein(-1)) to basal levels. Similar results were obtained with vinpocetine, and also with W-7. Combining 8-MM-IBMX and W-7 had no additive effect. To determine which PDE1 isoform is involved, we performed Western blot analysis for PDE1A, B, and C. Only Western blot analysis for PDE1C showed a characteristic band apparent at 80 kDa. Immunofluorescence showed cytoplasmic distribution of PDE1C and renin in the JG cells. In conclusion, PDE1C is expressed in isolated JG cells, and contributes to calcium's inhibitory modulation of renin release from JG cells.

Expression and function of the calcium-sensing receptor in juxtaglomerular cells.

Calcium-sensing receptors sense and translate micromolar changes of extracellular calcium into changes in intracellular calcium. Renin, a component of the renin-angiotensin system, is synthesized by, stored in, and released from the juxtaglomerular cells through a cAMP-dependent pathway. Increased intracellular calcium inhibits the adenylyl cyclase isoform type V, cAMP formation, and renin release from juxtaglomerular cells. We hypothesized that calcium-sensing receptors are expressed in juxtaglomerular cells and mediate changes in intracellular calcium and renin release. To test this we used primary cultures of isolated mouse juxtaglomerular cells in which we ran RT-PCR, Western blots, and immunofluorescence. RT-PCR showed a positive band at the expected 151 bp consistent with calcium-sensing receptor. Western blots showed a 130- to 150-kDa band confirming the calcium-sensing receptor in juxtaglomerular cells. Immunofluorescence and confocal microscopy using 2 different antibodies against the calcium-sensing receptor in juxtaglomerular cells showed positive fluorescence in the juxtaglomerular cells, which also had positive labeling for renin. To test whether calcium-sensing receptors regulate renin release, juxtaglomerular cells were incubated with a calcium-sensing receptor agonist, the calcimimetic cinacalcet-HCl, at concentrations of 50 and 1000 nmol/L in 0.25 mmol/L of calcium medium. Cinacalcet-HCl decreased juxtaglomerular cell cAMP formation to 47.3+/-6.8% and 44.2+/-9.7% of basal, respectively (P<0.001), and decreased renin release from 541.9+/-86.2 to 364.6+/-64.1 (P<0.05) and 279.6+/-56.9 (P<0.005) ng of angiotensin I per milliliter per hour per milligram of protein, respectively. We conclude that juxtaglomerular cells express the calcium-sensing receptor and that their activation leads to inhibition of adenylyl cyclase-V activity, decreasing cAMP formation and suppressing renin release.

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 micromol/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 microg 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 approximately 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.

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 micromol/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.