Abstract-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 mol/L of the cytosolic calcium chelator 5Ј5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,NЈ,NЈ-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Key Words: renin Ⅲ adenylyl cyclase Ⅲ calcium Ⅲ juxtaglomerular cell Ⅲ cAMP R enin 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][4][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.

Abstract-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. 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 ϩ2 ] 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 ϩ2 ] i could result in a cAMPmediated 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 calciuminhibitable isoform of adenylyl cyclase is in the JG cell and

Abstract-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. Key Words: renin Ⅲ calcium-sensing receptor Ⅲ cAMP Ⅲ calcium Ⅲ juxtaglomerular cells R enin is the critical enzyme in the formation of the potent vasoconstrictor angiotensin II. Renin is produced by, stored in, and released from juxtaglomerular (JG) cells, which are located in the lamina media of the afferent arteriole at the entrance to the glomerulus. 1 Renin secretion is controlled through a variety of regulatory pathways, 2 but all act through the second messenger cAMP, 3 the product of adenylyl cyclase activity. 4 It is well established that renin secretion by the JG cells, unlike most secretory cells, is inversely related to intracellular calcium concentration, such that paradoxically micromolar elevations in intracellular calcium are a potent inhibitor of renin release. [5][6][7][8] The nature of the interaction between cAMP and calcium has been resolved recently in that JG cAMP is synthesized by the calcium-inhibitable isoform of adenylyl cyclase type-V (AC-V), 9 -11 which exists within the JG cells, colocalized with the renin-containing granules. Thus, increased intracellular calcium will suppress AC-V activity and cAMP synthesis and retard the release of renin from the JG cells. 9,10 Although we now understand how intracellular calcium regulates cAMP formation and renin release, is not known how the JG cell modulates its intracellular calcium concentration or what signals drive this regulation.The calcium-sensing receptors (CaSRs) have a very important role in calcium homeostasis, regulating serum calcium via the secretion and action of parathyroid hormone and the excretion of calcium by the kidney in a negative feedback manner. 12 The CaSR is a G proteincoupled receptor of which the activation induces intracellular calcium oscillations by activating phospholipase C via a Gq protein, leading to the generation of inositol triphosphate. Inositol triphosphate releases calcium from intracellular stores in the endoplasmic reticulum. 13 In the kidney, the CaSR is known to regulate renal calcium excretion, influencing transepithelial movement of water and other electrolytes 14 in medullary and cortical thick ascending limb cells, the distal convoluted tubule, and cortical collecting duct cells, and both outer and inner

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 M) and vinpocetine (40 M)] and the calmodulin inhibitor W-7 (10 M) and measured cAMP and renin release. Stimulation of the JG cell CaSR with the calcimimetic cinacalcet (1 M) resulted in decreased cAMP from a basal of 1.13 Ϯ 0.14 to 0.69 Ϯ 0.08 pM/mg protein (P Ͻ 0.001) and in renin release from 0.89 Ϯ 0.16 to 0.38 Ϯ 0.08 g ANG I/ml ⅐ h Ϫ1 ⅐ mg protein Ϫ1 (P Ͻ 0.001). However, the addition of 8-MM-IBMX with cinacalcet returned both cAMP (1.10 Ϯ 0.19 pM/mg protein) and renin (0.57 Ϯ 0.16 g ANG I/ml ⅐ h Ϫ1 ⅐ mg protein Ϫ1 ) 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.angiotensin; calmodulin; phosphodiesterase; cAMP; adenylyl cyclase RENIN IS THE RATE-LIMITING enzymatic step in the production of angiotensin, a hormone that integrates cardiovascular and renal function in the control of blood pressure as well as salt and volume homeostasis (33). Renin is produced by, stored in, and released from juxtaglomerular (JG) cells, which are derived from renin progenitor cells (42) and are located in the afferent arteriole near the hilus of the glomerulus (2, 43, 35). Two main intracellular second messenger systems are known to regulate renin synthesis and secretion: the cyclic nucleotide, cyclic adenosine monophosphate (cAMP) (8, 41), and intracellular calcium (Ca) (21).Renin secretion by the JG cells demonstrates a paradoxical inverse relationship with intracellular calcium concentration, such that millimolar changes in extracellular Ca or micromolar elevations in intracellular calcium retard renin release (1,9,14,20,22). The primary second messenger involved in the regulation of renin release is cAMP (8), and the concentration of cAMP in cells is determined by a balance between the rate of cAMP generation by adenylyl cyclases and cAMP hydrolysis by phosphodiesterases (PDE) (7, 16). We (30, 31), and also Grünberger et al. (18), have reported JG cells express calciuminhibitable adenylyl cyclase (27). Furthermore, we have identified the JG-specific isoform-regulating renin release to be adenylyl cyclase type V (30)...

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.

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 Gq and/or Gi. Activation of Gq stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP3), releasing calcium from intracellular stores. Gi 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/IP3 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 ϭ 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 Ϯ 7% of control (P Ͻ 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 Ϯ 11% of control). The IP3 inhibitor 2-APB also reversed inhibition of renin from 56 Ϯ 6 to 104 Ϯ 11% of control (P Ͻ 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 Ϯ 8 to 118 Ϯ 22% of control (P Ͻ 0.01). Combining inhibition of IP3 and RyR was not additive. Gi inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 Ϯ 12 to 41 Ϯ 8% of control, P Ͻ 0.001). We conclude stimulating JG cell CaSR activates G q, initiating the PLC/IP3 pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition. calcium; renin; calcium-sensing receptor; inositol-3 phosphate; phospholipase C; protein kinase A; ryanodine receptor THE CALCIUM-SENSING RECEPTOR (CaSR) is a G protein-coupled 7-transmembrane receptor (33) found in the parathyroid gland, blood vessels, and kidney (18). In the parathyroid gland where CaSR was first described, high extracellular calcium results in decreased parathyroid hormone secretion (21). This is mediated by calcium activation of the CaSR, stimulating the G proteins G q and/or G i . In the parathyroid, activation of G q stimulates phospholipase C (PLC), producing diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ), the latter of which releases calcium from intracellular stores bound in the endoplasmic reticulum (ER) (48). G i stimulation leads to the inhibition of cAMP formation (16,48). Either pathway results in suppressing parathyroid hormone release.In the secretory juxtaglomerular (JG) cells, like the parathyroids, increased calcium results in the suppression of renin secretion (21,43,44). We have previously reported, both in vitro (44) and in vivo (6), that the JG cells contain CaSR, and that increased media calcium or activation of the CaSR with a calcimimetic (6, 43, 44) leads to suppression of renin secretion. JG cells synthesize, store, and release renin, the rate-limiting enzyme involved in the formation of angiotensin II (9). The cyclic nucleotide cAMP ...

Ortiz-Capisano MC, Atchison DK, Harding P, Lasley RD, Beierwaltes WH. Adenosine inhibits renin release from juxtaglomerular cells via an A 1 receptor-TRPC-mediated pathway. Am J Physiol Renal Physiol 305: F1209 -F1219, 2013. First published July 24, 2013 doi:10.1152/ajprenal.00710.2012.-Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A 1 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 A 1R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A 2R activation. Using primary cultures of isolated mouse JG cells, immunolabeling demonstrated renin and A 1R in JG cells, but not A2R subtypes, although RT-PCR indicated the presence of mRNA of both A 2AR 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 A 1R with 0.5 M N6-cyclohexyladenosine (CHA) decreased basal renin release from 0.22 Ϯ 0.05 to 0.14 Ϯ 0.03 g of angiotensin I generated per milliliter of sample per hour of incubation (AngI/ml/mg prot) (P Ͻ 0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35 Ϯ 0.08 g AngI/ml/mg prot; P Ͻ 0.01), and blocked renin inhibition by CHA (0.28 Ϯ 0.06 g AngI/ml/mg prot; P Ͻ 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 A 1R 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 A 1R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A 2 receptors do not regulate renin release. renin; adenosine; calcium; A 1R; CHA; adenosine receptors; transient receptor potential canonical 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: A 1 , A 2A , A 2B , and A 3 (13, 44). Adenosine inhibits renin secretion in vivo and in vitro (9, 37). It is considered that adenosine from the macula densa inhib...

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.