Bilateral renal cryodenervation decreases arterial pressure and improves insulin sensitivity in fructose-fed Sprague-Dawley rats.

Consumption of food high in fructose is prevalent in modern diets. One week of moderately high fructose intake combined with high salt diet has been shown to increase blood pressure and failed to suppress plasma renin activity (PRA). We tested the hypothesis that the hypertension and high PRA are consequences of elevated renal sympathetic nerve activity (RSNA). In protocol 1, we assessed RSNA by telemetry in conscious Sprague-Dawley rats given 20% fructose or 20% glucose in drinking water on a 0.4% NaCl diet (NS) for 1 wk and then transitioned to a 4% NaCl diet (HS). After an additional week, mean arterial pressure (MAP) and RSNA increased significantly in fructose-fed but not glucose-fed HS rats. In protocol 2, fructose (Fruc)- or glucose (Glu)-fed rats on NS or HS diet for 3 wk underwent sham denervation (shamDNX) or bilateral renal denervation using cryoablation (cryoDNX). MAP was higher in Fruc-HS rats compared with Glu-NS, Glu-HS, or Fruc-NS rats and decreased after cryoDNX ( P < 0.01). MAP did not change in Fruc-HS shamDNX rats. Renal norepinephrine content decreased by 85% in cryoDNX ( P < 0.01 vs. shamDNX). PRA significantly decreased after cryoDNX in both Fruc-NS and Fruc-HS rats. Nonfasting blood glucose levels were similar among the four groups. Glucose-to-insulin ratio significantly increased in Fruc-HS cryoDNX rats, consistent with greater insulin sensitivity. Taken together, these studies show that renal sympathoexcitation is, at least in part, responsible for salt-dependent increases in MAP, increased PRA, and decreased insulin sensitivity in rats fed a moderately high fructose diet for as little as 3 wk.

Salt restriction leads to activation of adult renal mesenchymal stromal cell-like cells via prostaglandin E2 and E-prostanoid receptor 4.

Despite the importance of juxtaglomerular cell recruitment in the pathophysiology of cardiovascular diseases, the mechanisms that underlie renin production under conditions of chronic stimulation remain elusive. We have previously shown that CD44+ mesenchymal-like cells (CD44+ cells) exist in the adult kidney. Under chronic sodium deprivation, these cells are recruited to the juxtaglomerular area and differentiate to new renin-expressing cells. Given the proximity of macula densa to the juxtaglomerular area and the importance of macula densa released prostanoids in renin synthesis and release, we hypothesized that chronic sodium deprivation induces macula densa release of prostanoids, stimulating renal CD44+ cell activation and differentiation. CD44+ cells were isolated from adult kidneys and cocultured with the macula densa cell line, MMDD1, in normal or low-sodium medium. Low sodium stimulated prostaglandin E2 production by MMDD1 and induced migration of CD44+ cells. These effects were inhibited by addition of a cyclooxygenase 2 inhibitor (NS398) or an E-prostanoid receptor 4 antagonist (AH23848) to MMDD1 or CD44+ cells, respectively. Addition of prostaglandin E2 to CD44+ cells increased cell migration and induced renin expression. In vivo activation of renal CD44+ cells during juxtaglomerular recruitment was attenuated in wild-type mice subjected to salt restriction in the presence of cyclooxygenase 2 inhibitor rofecoxib. Similar results were observed in E-prostanoid receptor 4 knockout mice subjected to salt restriction. These results show that the prostaglandin E2/E-prostanoid receptor 4 pathway plays a key role in the activation of renal CD44+ mesenchymal stromal cell-like cells during conditions of juxtaglomerular recruitment; highlighting the importance of this pathway as a key regulatory mechanism of juxtaglomerular recruitment.

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.

The influence of extracellular and intracellular calcium on the secretion of renin.

Changes in plasma, extracellular, and intracellular calcium can affect renin secretion from the renal juxtaglomerular (JG) cells. Elevated intracellular calcium directly inhibits renin release from JG cells by decreasing the dominant second messenger intracellular cyclic adenosine monophosphate (cAMP) via actions on calcium-inhibitable adenylyl cyclases and calcium-activated phosphodiesterases. Increased extracellular calcium also directly inhibits renin release by stimulating the calcium-sensing receptor (CaSR) on JG cells, resulting in parallel changes in the intracellular environment and decreasing intracellular cAMP. In vivo, acutely elevated plasma calcium inhibits plasma renin activity (PRA) via parathyroid hormone-mediated elevations in renal cortical interstitial calcium that stimulate the JG cell CaSR. However, chronically elevated plasma calcium or CaSR activation may actually stimulate PRA. This elevation in PRA may be a compensatory mechanism resulting from calcium-mediated polyuria. Thus, changing the extracellular calcium in vitro or in vivo results in inversely related acute changes in cAMP, and therefore renin release, but chronic changes in calcium may result in more complex interactions dependent upon the duration of changes and the integration of the body's response to these changes.

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.

Parathyroid hormone stimulates juxtaglomerular cell cAMP accumulation without stimulating renin release.

Parathyroid hormone (PTH) is positively coupled to the generation of cAMP via its actions on the PTH1R and PTH2R receptors. Renin secretion from juxtaglomerular (JG) cells is stimulated by elevated intracellular cAMP, and every stimulus that increases renin secretion is thought to do so via increasing cAMP. Thus we hypothesized that PTH increases renin release from primary cultures of mouse JG cells by elevating intracellular cAMP via the PTH1R receptor. We found PTH1R, but not PTH2R, mRNA expressed in JG cells. While PTH increased JG cell cAMP content from (log(10) means ± SE) 3.27 ± 0.06 to 3.92 ± 0.12 fmol/mg protein (P < 0.001), it did not affect renin release. The PTH1R-specific agonist, parathyroid hormone-related protein (PTHrP), also increased JG cell cAMP from 3.13 ± 0.09 to 3.93 ± 0.09 fmol/mg protein (P < 0.001), again without effect on renin release. PTH2R receptor agonists had no effect on cAMP or renin release. PTHrP increased cAMP in the presence of both low and high extracellular calcium from 3.31 ± 0.17 to 3.83 ± 0.20 fmol/mg protein (P < 0.01) and from 3.29 ± 0.18 to 3.63 ± 0.22 fmol/mg protein (P < 0.05), respectively, with no effect on renin release. PTHrP increased JG cell cAMP in the presence of adenylyl cyclase-V inhibition from 2.85 ± 0.17 to 3.44 ± 0.14 fmol/mg protein (P < 0.001) without affecting renin release. As a positive control, forskolin increased JG cell cAMP from 3.39 ± 0.13 to 4.48 ± 0.07 fmol/mg protein (P < 0.01) and renin release from 2.96 ± 0.10 to 3.29 ± 0.08 ng ANG I·mg prot(-1)·h(-1) (P < 0.01). Thus PTH increases JG cell cAMP via non-calcium-sensitive adenylate cyclases without affecting renin release. These data suggest compartmentalization of cAMP signaling in JG cells.

Parathyroid hormone-related protein stimulates plasma renin activity via its anorexic effects on sodium chloride intake.

Parathyroid hormone-related protein (PTHrP) increases renin release from isolated perfused kidneys and may act as an autacoid regulator of renin secretion, but its effects on renin in vivo are unknown. In vivo, PTHrP causes hypercalcemia and anorexia, which may affect renin. We hypothesized that chronically elevated PTHrP would increase plasma renin activity (PRA) indirectly via its anorexic effects, reducing sodium chloride (NaCl) intake and causing NaCl restriction. We infused male Sprague-Dawley rats with the vehicle (control) or 125 µg PTHrP/day (PTHrP) via subcutaneous osmotic minipumps for 5 days. To replenish NaCl consumption, a third group of PTHrP-infused rats received 0.3% NaCl (PTHrP + NaCl) in their drinking water. PTHrP increased PRA from a median control value of 3.68 to 18.4 ng Ang I·ml(-1)·h(-1) (P < 0.05), whereas the median PTHrP + NaCl PRA value was normal (7.82 ng Ang I·ml(-1)·h(-1), P < 0.05 vs. PTHrP). Plasma Ca(2+) (median control: 10.2 mg/dl; PTHrP: 13.7 mg/dl; PTHrP + NaCl: 14.1 mg/dl; P < 0.05) and PTHrP (median control: 0.03 ng/ml; PTHrP: 0.12 ng/ml; PTHrP + NaCl: 0.15 ng/ml; P < 0.05) were elevated in PTHrP- and PTHrP + NaCl-treated rats. Body weights and caloric consumption were lower in PTHrP- and PTHrP + NaCl-treated rats. NaCl consumption was lower in PTHrP-treated rats (mean Na(+): 28.5 ± 4.1 mg/day; mean Cl(-): 47.8 mg/day) compared with controls (Na(+): 67.3 ± 2.7 mg/day; Cl(-): 112.8 ± 4.6 mg/day; P < 0.05). NaCl consumption was comparable with control in the PTHrP + NaCl group; 0.3% NaCl in the drinking water had no effect on PRA in normal rats. Thus, our data support the hypothesis that PTHrP increases PRA via its anorexic effects, reducing NaCl intake and causing NaCl restriction.

Renovascular hypertension using a modified two-kidney, one-clip approach in mice is not dependent on the α1 or α2 Na-K-ATPase ouabain-binding site.

Endogenous cardiotonic steroids, through their interaction with the ouabain-binding site of the Na-K-ATPase α-subunit, have been implicated in a variety of cardiovascular disease states including hypertension. We have previously shown that ACTH-induced hypertension is abolished in mutant mice expressing ouabain-resistant α1- and α2-subunits. To further evaluate hypertension resistance in these mutant mice, we examined blood pressure changes in a modified model of 2-kidney, 1-clip (2K1C) renovascular hypertension. To reliably generate 2K1C hypertension, we used polyvinyl tubing (inner diameter: ∼0.27 mm) to accurately gauge the degree of renal artery stenosis. Using this method, virtually all of the clipped mice became hypertensive and there was no incidence of apparent renal ischemia. By telemetry, in response to renal artery clipping, blood pressure in wild-type mice (α1 ouabain-resistant, α2 ouabain-sensitive) increased from 97 ± 3 to 136 ± 7 mmHg. In α1-resistant, α2-resistant mice, pressure increased from 93 ± 2 to 123 ± 4 mmHg, and in α1-sensitive, α2-resistant mice, blood pressure increased from 95 ± 2 to 139 ± 5 mmHg. Blood pressure changes were equivalent in all three groups. In sham mice, blood pressure did not change (96 ± 1 to 95 ± 2 mmHg). Renin mRNA expression was dramatically elevated in the left vs. the right kidney, and plasma renin concentration was elevated similarly in all genotypes. These data indicate that sensitivity of the α1- or α2-Na-K-ATPase binding site to cardiotonic steroids is not a prerequisite for the development of 2K1C renovascular hypertension. In addition, use of a polyurethane cuff to constrict the renal artery provides a reliable method for producing 2K1C hypertension in mice.

The role of calcium in the regulation of renin secretion.

Renin is the enzyme which is the rate-limiting step in the formation of the hormone angiotensin II. Therefore, the regulation of renin secretion is critical in understanding the control of the renin-angiotensin-aldosterone system and its many biological and pathological actions. Renin is synthesized, stored in, and released from the juxtaglomerular (JG) cells of the kidney. While renin secretion is positively regulated by the "second messenger" cAMP, unlike most secretory cells, renin secretion from the JG cell is inversely related to the extracellular and intracellular calcium concentrations. This novel relationship is referred to as the "calcium paradox." This review will address observations made over the past 30 years regarding calcium and the regulation of renin secretion, and focus on recent observations which address this scientific conundrum. These include 1) receptor-mediated pathways for changing intracellular calcium; 2) the discovery of a calcium-inhibitable isoform of adenylyl cyclase associated with renin in the JG cells; 3) calcium-sensing receptors in the JG cells; 4) calcium-calmodulin-mediated signals; 5) the role of phosphodiesterases; and 6) connexins, gap junctions, calcium waves, and the cortical extracellular calcium environment. While cAMP is the dominant second messenger for renin secretion, calcium appears to modulate the integrated activities of the enzymes, which balance cAMP synthesis and degradation. Thus this review concludes that calcium modifies the amplitude of cAMP-mediated renin-signaling pathways. While calcium does not directly control renin secretion, increased calcium inhibits and decreased calcium amplifies cAMP-stimulated renin secretion.

Decreased renal perfusion rapidly increases plasma membrane Na-K-ATPase in rat cortex by an angiotensin II-dependent mechanism.

To understand how rapid changes in blood pressure can regulate Na-K-ATPase in the kidney cortex, we tested the hypothesis that a short-term (5 min) decrease in renal perfusion pressure will increase the amount of Na-K-ATPase in the plasma membranes by an angiotensin II-dependent mechanism. The abdominal aorta of anesthetized Sprague-Dawley rats was constricted with a ligature between the renal arteries, and pressure was monitored on either side during acute constriction. Left renal perfusion pressure was reduced to 70 +/- 1 mmHg (n = 6), whereas right renal perfusion pressure was 112 +/- 4 mmHg. In control (nonconstricted) rats (n = 5), pressure to both kidneys was similar at 119 +/- 6 mmHg. After 5 min of reduced perfusion, femoral venous samples were taken for plasma renin activity (PRA) and the kidneys excised. The cortex was dissected, minced, sieved, and biotinylated. Lower perfusion left kidneys showed a 41% increase (P < 0.003) in the amount of Na-K-ATPase in the plasma membrane compared with right kidneys. In controls, there was no difference in cell surface Na-K-ATPase between left and right kidneys (P = 0.47). PRA was 57% higher in experimental animals compared with controls. To test the role of angiotensin II in mediating the increase in Na-K-ATPase, we repeated the experiments (n = 6) in rats treated with ramiprilat. When angiotensin-converting enzyme was inhibited, the cell surface Na-K-ATPase of the two kidneys was equal (P =0.46). These results confirm our hypothesis: rapid changes in blood pressure regulate trafficking of Na-K-ATPase in the kidney cortex.

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.

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.

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.

cGMP stimulates renin secretion in vivo by inhibiting phosphodiesterase-3.

The interaction between renin, nitric oxide (NO), and its second messenger cGMP is controversial. cAMP is the stimulatory second messenger for renin but is degraded by phosphodiesterases (PDEs). We previously reported that increasing endogenous cGMP in rats by inhibiting its breakdown by PDE-5 stimulated renin secretion rate (RSR). This could be reversed by selective inhibition of neuronal nitric oxide synthase (nNOS). PDE-3 metabolizes cAMP, but this can be inhibited by cGMP, suggesting that renal cGMP could stimulate RSR by diminishing PDE-3 degradation of cAMP. Rats were anesthetized with Inactin before determination of blood pressure (BP), renal blood flow (RBF), and sampling of renal venous and arterial blood to determine RSR. In 13 rats, basal BP was 104 +/- 2 mmHg, RBF was 6.1 ml x min(-1) x g kidney wt(-1) and RSR was 2.9 +/- 1.4 ng ANG I x h(-1) x min(-1). Inhibiting PDE-5 with 20 mg/kg body wt i.p. Zaprinast did not change hemodynamic parameters but increased RSR fivefold to 12.2 +/- 4.9 ng ANG I x h(-1) x min(-1) (P < 0.05). Renal venous cAMP was increased by Zaprinast from 93.8 +/- 27.9 to 149.2 +/- 36.0 pM x min(-1) x g kidney wt(-1) (P < 0.05). When another 10 rats were treated with the PDE-3 inhibitor Milrinone (0.4 microg/min over 30 min, which did not affect hemodynamics), RSR was elevated to 10.4 +/- 4.4 ng ANG I x h(-1) x min(-1). Milrinone also increased renal venous cAMP from 212 +/- 29 to 304 +/- 29 pM x min(-1) x g kidney wt(-1) (P < 0.025). Administration of Zaprinast to rats pretreated with Milrinone (n = 10) did not further increase in RSR (7.5 +/- 3.3 ng ANG I x h(-1) x min(-1)). These results are consistent with endogenous renal cGMP inhibiting PDE-3, which diminishes renal metabolism of cAMP. The resulting increase in cAMP serves as an endogenous stimulus for renin secretion. This suggests a pathway by which NO can indirectly stimulate RSR through its second messenger cGMP.

Extracellular ATP stimulates NO production in rat thick ascending limb.

NO produced by NO synthase (NOS) 3 acts as an autacoid to regulate NaCl absorption in the thick ascending limb. ATP induces NO production by NOS 3 in endothelial cells. We hypothesized that extracellular ATP activates NOS in thick ascending limbs through P2 receptors. To test this, we measured intracellular NO production using the NO-selective fluorescent dye DAF-2 in suspensions of rat medullary thick ascending limbs. We found that ATP increased DAF-2 fluorescence in a concentration-dependent manner, reaching saturation at &200 micromol/L with an EC50 of 37 micromol/L. The increase was blunted by 74% by the nonselective NOS inhibitor L-omega-nitro-arginine-methyl-ester (2 mmol/L; 60+/-7 versus 16+/-6 arbitrary fluorescence units; P<0.02; n=5). In the presence of the P2 receptor antagonist suramin (300 micromol/L), ATP-induced NO production was reduced by 64% (101+/-11 versus 37+/-5 arbitrary fluorescence units; P<0.002; n=5). Blocking ATP hydrolysis with a 5'-ectonucleotidase inhibitor, ARL67156 (30 micromol/L) enhanced the response to ATP and shifted the EC(50) to 0.8 micromol/L. In the presence of ARL67156, the EC50 of the P2X-selective agonist beta,gamma-methylene-adenosine 5'-triphosphate was 4.8 micromol/L and the EC50 for the P2Y-selective agonist UTP was 40.4 micromol/L. The maximal responses for both agonists were similar. Taken together, these data indicate that ATP stimulates NO production in the thick ascending limb primarily through P2X receptor activation and that ATP hydrolysis may regulate NO production.