8-Aminoguanine and Its Actions on Renal Excretory Function.

BACKGROUND: The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. METHODS: Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. RESULTS: Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. CONCLUSIONS: 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.

Biochemical pathways of 8-aminoguanine production in Sprague-Dawley and Dahl salt-sensitive rats.

BACKGROUND: 8-Aminoguanine exerts natriuretic and antihypertensive activity. Whether and how "free" 8-aminoguanine exists in vivo is unclear. Because 8-nitroguanosine is naturally occurring, we tested the hypothesis that 8-aminoguanine can arise from: pathway 1, 8-nitroguanosine â†’ 8-aminoguanosine â†’ 8-aminoguanine; and pathway 2, 8-nitroguanosine â†’ 8-nitroguanine â†’ 8-aminoguanine. METHODS: 8-Aminoguanine biosynthesis was explored in rats using renal microdialysis, mass spectrometry and enzyme kinetics. RESULTS: In Sprague-Dawley rats, 8-nitroguanosine infusions increased kidney levels of 8-nitroguanine, 8-aminoguanosine and 8-aminoguanine; 8-nitroguanine infusions increased 8-aminoguanine. Purine nucleoside phosphorylase (PNPase) converted 8-nitroguanosine to 8-nitroguanine and 8-aminoguanosine to 8-aminoguanine. Forodesine (PNPase inhibitor) reduced metabolism of 8-nitroguanosine by pathway 2 and shunted metabolism of 8-nitroguanosine to 8-aminoguanosine. In Dahl salt-sensitive rats, 8-nitroguanosine infusions increased kidney levels of 8-nitroguanine, 8-aminoguanosine and 8-aminoguanine. These results indicate that both pathways 1 and 2 participate in the biosynthesis of 8-aminoguanine in Sprague-Dawley and Dahl rats. Endogenous 8-aminoguanine in kidneys and urine were elevated many-fold in Dahl, compared to Sprague-Dawley, rats. The increased levels of 8-aminoguanine in Dahl rats were not due to alterations in pathways 1 and 2 but were associated with increased urine levels of endogenous 8-nitroguanosine suggesting that the "upstream" production of 8-nitroguanosine was increased in Dahl rats. Dahl rats are known to have high levels of peroxynitrite, and peroxynitrite is known to nitrate guanosine in biomolecules. Here we confirm that a peroxynitrite donor increases kidney levels of 8-aminoguanine. CONCLUSION: 8-Aminoguanine occurs naturally via two distinct pathways and kidney levels of 8-aminoguanine are increased in Dahl rats, likely due to increased production of 8-nitroguanosine, a by-product of peroxynitrite chemistry.

Long-Term Dipeptidyl Peptidase 4 Inhibition Worsens Hypertension and Renal and Cardiac Abnormalities in Obese Spontaneously Hypertensive Heart Failure Rats.

Background The long-term effects of dipeptidyl peptidase 4 (DPP4) inhibitors on blood pressure and cardiovascular and renal health remain controversial. Herein, we investigated the extended (>182 days) effects of DPP4 inhibition in a model of spontaneous hypertension, heart failure, diabetes mellitus, obesity and hyperlipidemia. Methods and Results Adult obese spontaneously hypertensive heart failure rats (SHHF) were implanted with radio transmitters for measurement of arterial blood pressures. Two weeks later, SHHF were randomized to receive either a DPP4 inhibitor (sitagliptin, 80 mg/kg per day in drinking water) or placebo. At the end of the radiotelemetry measurements, renal and cardiac function and histology, as well as other relevant biochemical parameters, were assessed. For the first 25 days, mean arterial blood pressures were similar in sitagliptin-treated versus control SHHF; afterwards, mean arterial blood pressures increased more in sitagliptin-treated SHHF (P<0.000001). The time-averaged mean arterial blood pressures from day 26 through 182 were 7.2 mm Hg higher in sitagliptin-treated SHHF. Similar changes were observed for systolic (8.6 mm Hg) and diastolic (6.1 mm Hg) blood pressures, and sitagliptin augmented hypertension throughout the light-dark cycle. Long-term sitagliptin treatment also increased kidney weights, renal vascular resistances, the excretion of kidney injury molecule-1 (indicates injury to proximal tubules), renal interstitial fibrosis, glomerulosclerosis, renal vascular hypertrophy, left ventricular dysfunction, right ventricular degeneration, and the ratios of collagen IV/collagen III and collagen IV/laminin in the right ventricle. Conclusions These findings indicate that, in some genetic backgrounds, long-term DPP4 inhibitor treatment is harmful and identify an animal model to study mechanisms of, and test ways to prevent, DPP4 inhibitor-induced pathological conditions.

Brief Report: Dipyridamole Decreases Gut Mucosal Regulatory T-Cell Frequencies Among People With HIV on Antiretroviral Therapy.

BACKGROUND: We had previously conducted a double-blind, randomized placebo-controlled, partial cross-over trial showing that 12 weeks of dipyridamole decreased CD8 T-cell activation among treated HIV(+) individuals by increasing extracellular adenosine levels. METHODS: In this substudy, rectosigmoid biopsies were obtained from 18 participants (9 per arm), to determine whether 12 weeks of dipyridamole affects mucosal immune cells. Participants randomized to placebo were then switched to dipyridamole for 12 weeks while the treatment arm continued dipyridamole for another 12 weeks. We evaluated T-cell frequencies and plasma markers of microbial translocation and intestinal epithelial integrity. Linear regression models on log-transformed outcomes were used for the primary 12-week analysis. RESULTS: Participants receiving dipyridamole had a median 70.2% decrease from baseline in regulatory T cells (P = 0.007) and an 11.3% increase in CD8 T cells (P = 0.05). There was a nonsignificant 10.80% decrease in plasma intestinal fatty acid binding protein levels in the dipyridamole arm compared with a 9.51% increase in the placebo arm. There were no significant differences in plasma levels of ß-D-glucan. In pooled analyses, there continued to be a significant decrease in regulatory T cells (-44%; P = 0.004). There was also a trend for decreased CD4 and CD8 T-cell activation. CONCLUSION: Increasing extracellular adenosine levels using dipyridamole in virally suppressed HIV (+) individuals on antiretroviral therapy can affect regulation of gut mucosal immunity.

Author Correction: Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2',3'-cAMP.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Purine Metabolites in Tumor-Derived Exosomes May Facilitate Immune Escape of Head and Neck Squamous Cell Carcinoma.

Body fluids of patients with head and neck squamous cell carcinoma (HNSCC) are enriched in exosomes that reflect properties of the tumor. The aim of this study was to determine whether purine metabolites are carried by exosomes and evaluate their role as potential contributors to tumor immune escape. The gene expression levels of the purine synthesis pathway were studied using the Cancer Genome Atlas (TCGA) Head and Neck Cancer database. Exosomes were isolated from supernatants of UMSCC47 cells and from the plasma of HNSCC patients (n = 26) or normal donors (NDs; n = 5) using size exclusion chromatography. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to assess levels of 19 purine metabolites carried by exosomes. In HNSCC tissues, expression levels of genes involved in the purinergic pathway were upregulated indicating an accelerated purine metabolism compared to normal tissues. Exosomes from supernatants of UMSCC47 cells contained several purine metabolites, predominantly adenosine and inosine. Purine metabolite levels were enriched in exosomes isolated from the plasma of HNSCC patients compared to those isolated from NDs and carried elevated levels of adenosine (p = 0.0223). Exosomes of patients with early-stage disease and no lymph node metastasis contained significantly elevated levels of adenosine and 5'-GMP (p = 0.0247 and p = 0.0229, respectively). The purine metabolite levels in exosomes decreased in patients with advanced cancer and nodal involvement. This report provides the first evidence that HNSCC cells shuttle purine metabolites in exosomes, with immunosuppressive adenosine being the most prominent purine. Changes in the content and levels of purine metabolites in circulating exosomes reflect disease progression in HNSCC patients.

Tumor-derived exosomes promote angiogenesis via adenosine A2B receptor signaling.

RATIONALE: One hallmark of tumor-derived exosomes (TEX) is the promotion of cancer progression by stimulating angiogenesis. This study was performed to evaluate the role of adenosine receptors in TEX-induced angiogenesis. METHODS: TEX produced by UMSCC47 head and neck cancer cell line were isolated by mini size exclusion chromatography (mini-SEC). Enzymatic activity of ectonucleotidases CD39/CD73 carried by TEX was measured by HPLC. Adenosine content of TEX was measured by UPLC-MS/MS. Primary human macrophages were co-incubated with TEX or exosomes derived from the plasma of head and neck cancer patients and their marker expression profile was analyzed by flow cytometry. The macrophage secretome was analyzed by angiogenesis arrays. The in vitro angiogenic potential of TEX was evaluated in endothelial growth studies. Results were validated in vivo using basement membrane extract plug assays in A1R-/-, A2AR-/- and A2BR-/- rats. Vascularization was analyzed by hemoglobin quantification and immunohistology with vessel and macrophage markers. RESULTS: TEX carried enzymatically active CD39/CD73 and adenosine. TEX promoted A2BR-mediated polarization of macrophages toward an M2-like phenotype (p < 0.05) and enhanced their secretion of angiogenic factors. Growth of endothelial cells was stimulated directly by TEX and indirectly via macrophage-reprogramming dependent on A2BR signaling (p < 0.01). In vivo, TEX stimulated the formation of defined vascular structures and macrophage infiltration. This response was absent in A2BR-/- rats (p < 0.05). CONCLUSION: This report provides the first evidence for adenosine production by TEX to promote angiogenesis via A2BR. A2BR antagonism emerges as a potential strategy to block TEX-induced angiogenesis.

Adenosine receptors regulate exosome production.

Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A1Rs, A2ARs, and A2BRs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A1R-/-, A2AR-/-, and A2BR-/- rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A1Rs, A2ARs, and A2BRs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A1R and A2AR constrained exosome production under normal conditions (p = 0.0297; p = 0.0409, respectively), and A1R, A2AR, and A2BR constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p = 0.0028) by the selective A2AR agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A1R and A2BR agonists (p = 0.0474). The selective A2AR antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A2BR antagonist MRS 1754 reduced exosome production. Our findings indicate that A2ARs suppress exosome release in all cell types examined, whereas effects of A1Rs and A2BRs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A2AR antagonists may inadvertently increase exosome production from tumor cells.

Characterization of the N6-etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism.

The goal of this study was to determine the validity of using N6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N6-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N6-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-nucleotidases process N6-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66 h) metabolized N6-etheno-ATP. Applied N6-etheno-ATP was recovered in the medium as N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and surprisingly N6-etheno-adenine; intracellular N6-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N6-etheno-ATP, N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and N6-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors (3H-α,ß-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (35S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N6-etheno-adenosine was partially converted to N6-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N6-etheno-ATP was quickly metabolized, with N6-etheno-adenine being the main product in naïve rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N6-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N6-etheno-adenosine to N6-etheno-adenine.

Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2'3'-cAMP.

Exosome secretion by cells is a complex, poorly understood process. Studies of exosomes would be facilitated by a method for increasing their production and release. Here, we present a method for stimulating the secretion of exosomes. Cultured cells were treated or not with sodium iodoacetate (IAA; glycolysis inhibitor) plus 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor). Exosomes were isolated by size-exclusion chromatography and their morphology, size, concentration, cargo components and functional activity were compared. IAA/DNP treatment (up to 10 µM each) was non-toxic and resulted in a 3 to 16-fold increase in exosome secretion. Exosomes from IAA/DNP-treated or untreated cells had similar biological properties and functional effects on endothelial cells (SVEC4-10). IAA/DNP increased exosome secretion from mouse organ cultures, and in vivo injections enhanced the levels of circulating exosomes. IAA/DNP decreased ATP levels (p < 0.05) in cells. A cell membrane-permeable form of 2',3'-cAMP and 3'-AMP mimicked the potentiating effects of IAA/DNP on exosome secretion. In cells lacking 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; an enzyme that metabolizes 2',3'-cAMP into 2'- and 3'-AMP), effects of IAA/DNP on exosome secretion were enhanced. The IAA/DNP combination is a powerful stimulator of exosome secretion, and these stimulatory effects are, in part, mediated by intracellular 2',3'-cAMP.

DPP4 Inhibition, NPY1-36, PYY1-36, SDF-1α, and a Hypertensive Genetic Background Conspire to Augment Cell Proliferation and Collagen Production: Effects That Are Abolished by Low Concentrations of 2-Methoxyestradiol.

By reducing their metabolism, dipeptidyl peptidase 4 inhibition (DPP4I) enhances the effects of numerous peptides including neuropeptide Y1-36 (NPY1-36), peptide YY1-36 (PYY1-36), and SDF-1α Studies show that separately NPY1-36, PYY1-36 and SDF-1α stimulate proliferation of, and collagen production by, cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), and glomerular mesangial cells (GMCs), particularly in cells isolated from genetically hypertensive rats. Whether certain combinations of these factors, in the absence or presence of DPP4I, are more profibrotic than others is unknown. Here we contrasted 24 different combinations of conditions (DPP4I, hypertensive genotype and physiologic levels [3 nM] of NPY1-36, PYY1-36, or SDF-1α) on proliferation of, and [3H]-proline incorporation by, CFs, PGVSMCs, and GMCs. In all three cell types, the various treatment conditions differentially increased proliferation and [3H]-proline incorporation, with a hypertensive genotype + DPP4I + NPY1-36 + SDF-1α being the most efficacious combination. Although the effects of this four-way combination were similar in male versus female CFs, physiologic (1 nM) concentrations of 2-methoxyestradiol (2ME; nonestrogenic metabolite of 17ß-estradiol), abolished the effects of this combination in both male and female CFs. In conclusion, this study demonstrates that CFs, PGVSMCs, and GMCs are differentially activated by various combinations of NPY1-36, PYY1-36, SDF-1α, a hypertensive genetic background and DPP4I. We hypothesize that as these progrowth conditions accumulate, a tipping point would be reached that manifests in the long term as organ fibrosis and that 2ME would obviate any profibrotic effects of DPP4I, even under the most profibrotic conditions (i.e., hypertensive genotype with high NPY1-36 + SDF-1α levels and low 2ME levels). SIGNIFICANCE STATEMENT: This work elucidates combinations of factors that could contribute to long-term profibrotic effects of dipeptidyl peptidase 4 inhibitors and suggests a novel drug combination that could prevent any potential profibrotic effects of dipeptidyl peptidase 4 inhibitors while augmenting the protective effects of this class of antidiabetic agents.

A Randomized, Placebo-Controlled, Pilot Clinical Trial of Dipyridamole to Decrease Human Immunodeficiency Virus-Associated Chronic Inflammation.

BACKGROUND: Adenosine is a potent immunoregulatory nucleoside produced during inflammatory states to limit tissue damage. We hypothesized that dipyridamole, which inhibits cellular adenosine uptake, could raise the extracellular adenosine concentration and dampen chronic inflammation associated with human immunodeficiency virus (HIV) type 1. METHODS: Virally suppressed participants receiving antiretroviral therapy were randomized 1:1 for 12 weeks of dipyridamole (100 mg 4 times a day) versus placebo capsules. All participants took open-label dipyridamole during weeks 12-24. Study end points included changes in markers of systemic inflammation (soluble CD163 and CD14, and interleukin 6) and levels of T-cell immune activation (HLA-DR+CD38+). RESULTS: Of 40 participants who were randomized, 17 dipyridamole and 18 placebo recipients had baseline and week 12 data available for analyses. There were no significant changes in soluble markers, apart from a trend toward decreased levels of soluble CD163 levels (P = .09). There was a modest decrease in CD8+ T-cell activation (-17.53% change for dipyridamole vs +13.31% for placebo; P = .03), but the significance was lost in the pooled analyses (P = .058). Dipyridamole also reduced CD4+ T-cell activation (-11.11% change; P = .006) in the pooled analyses. In post hoc analysis, detectable plasma dipyridamole levels were associated with higher levels of inosine, an adenosine surrogate, and of cyclic adenosine monophosphate. CONCLUSION: Dipyridamole increased extracellular adenosine levels and decreased T-cell activation significantly among persons with HIV-1 infection receiving virally suppressive therapy.

Adenosine, Via A2B Receptors, Inhibits Human (P-SMC) Progenitor Smooth Muscle Cell Growth.

c-Kit+ progenitor smooth muscle cells (P-SMCs) can develop into SMCs that contribute to injury-induced neointimal thickening. Here, we investigated whether adenosine reduces P-SMC migration and proliferation and whether this contributes to adenosine's inhibitory actions on neointima formation. In human P-SMCs, 2-chloroadenosine (stable adenosine analogue) and BAY60-6583 (A2B agonist) inhibited P-SMC proliferation and migration. Likewise, increasing endogenous adenosine by blocking adenosine metabolism with erythro-9-(2-hydroxy-3-nonyl) adenine (inhibits adenosine deaminase) and 5-iodotubercidin (inhibits adenosine kinase) attenuated P-SMC proliferation and migration. Neither N6-cyclopentyladenosine (A1 agonist), CGS21680 (A2A agonist), nor N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (A3 agonist) affected P-SMC proliferation or migration. 2-Chloroadenosine increased cyclic AMP, reduced Akt phosphorylation (activates cyclin D expression), and reduced levels of cyclin D1 (promotes cell-cycle progression). Moreover, 2-chloroadenosine inhibited expression of Skp2 (promotes proteolysis of p27Kip1) and upregulated levels of p27Kip1 (negative cell-cycle regulator). A2B receptor knockdown prevented the effects of 2-chloroadenosine on cyclic AMP production and P-SMC proliferation and migration. Likewise, inhibition of adenylyl cyclase and protein kinase A rescued P-SMCs from the inhibitory effects of 2-chloroadenosine. The inhibitory effects of adenosine were similar in male and female P-SMCs. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 µmol/L for 7 days) reduced neointimal hyperplasia by 64.5% (P<0.05; intima/media ratio: control, 1.4±0.02; treated, 0.53±0.012) and reduced neointimal c-Kit+ cells. Adenosine inhibits P-SMC migration and proliferation via the A2B receptor/cyclic AMP/protein kinase A axis, which reduces cyclin D1 expression and activity via inhibiting Akt phosphorylation and Skp2 expression and upregulating p27kip1 levels. Adenosine attenuates neointima formation in part by inhibiting infiltration and proliferation of c-Kit+ P-SMCs.

Extracellular Ubiquitin(1-76) and Ubiquitin(1-74) Regulate Cardiac Fibroblast Proliferation.

SDF-1α (stromal cell-derived factor-1α) is a CXCR4-receptor agonist and DPP4 (dipeptidyl peptidase 4) substrate. SDF-1α, particularly when combined with sitagliptin to block the metabolism of SDF-1α by DPP4, stimulates proliferation of cardiac fibroblasts via the CXCR4 receptor; this effect is greater in cells from spontaneously hypertensive rats versus Wistar-Kyoto normotensive rats. Emerging evidence indicates that ubiquitin(1-76) exists in plasma and is a potent CXCR4-receptor agonist. Therefore, we hypothesized that ubiquitin(1-76), similar to SDF-1α, should increase proliferation of cardiac fibroblasts. Contrary to our working hypothesis, ubiquitin(1-76) did not stimulate cardiac fibroblast proliferation, yet unexpectedly antagonized the proproliferative effects of SDF-1α combined with sitagliptin. In this regard, ubiquitin(1-76) was more potent in spontaneously hypertensive versus Wistar-Kyoto cells. In the presence of 6bk (selective inhibitor of insulin-degrading enzyme [IDE]; an enzyme known to convert ubiquitin(1-76) to ubiquitin(1-74)), ubiquitin(1-76) no longer antagonized the proproliferative effects of SDF-1α/sitagliptin. Ubiquitin(1-74) also antagonized the proproliferative effects of SDF-1α/sitagliptin, and this effect of ubiquitin(1-74) was not blocked by 6bk and was >10-fold more potent compared with ubiquitin(1-76). Neither ubiquitin(1-76) nor ubiquitin(1-74) inhibited the proproliferative effects of the non-CXCR4 receptor agonist neuropeptide Y (activates Y1 receptors). Cardiac fibroblasts expressed IDE mRNA, protein, and activity and converted ubiquitin(1-76) to ubiquitin(1-74). Spontaneously hypertensive fibroblasts expressed greater IDE activity. Extracellular ubiquitin(1-76) blocks the proproliferative effects of SDF-1α/sitagliptin via its conversion by IDE to ubiquitin(1-74), a potent CXCR4 antagonist. Thus, IDE inhibitors, particularly when combined with DPP4 inhibitors or hypertension, could increase the risk of cardiac fibrosis.

Adenosine Receptors Influence Hypertension in Dahl Salt-Sensitive Rats: Dependence on Receptor Subtype, Salt Diet, and Sex.

The influence of adenosine receptors on blood pressure in salt-sensitive hypertension is unknown. Here, we examined the effects of salt diets on arterial blood pressures (radiotelemetry) in female and male Dahl salt-sensitive wild-type versus female and male Dahl salt-sensitive A1, A2A, or A2B receptor knockouts (A1KOs, A2AKOs, and A2BKOs, respectively). At baseline, all rats were on a 0.3% salt diet; then separate groups were switched to either 4% or 8% salt diet for 2 weeks. Compared with wild-types, baseline pressures were not affected by knockout of A1 or A2B receptors; yet, mean, systolic, and diastolic pressures were significantly (P<0.01) higher in A2AKOs versus wild-types, an effect independent of sex. During the second week on a 4% salt diet, mean, systolic, and diastolic blood pressures (mm Hg, mean±SEM) in female A1KOs (176±5, 209±5, and 147±4, respectively) and A2BKOs (166±8, 198±9, and 139±8, respectively) were significantly lower (P<0.001) than wild-type on a 4% salt diet (202±4, 240±5, and 172±3, respectively). Male A1KOs and A2BKOs were not protected against 4% salt diet-induced hypertension. This female advantage was overwhelmed by an 8% salt diet. Female and male A2AKOs were more salt sensitive, a phenotype that was apparent in male A2AKOs on 4% and 8% salt diets and in females on 8% salt diet. Female A1KOs and A2BKOs were less susceptible to salt-induced stroke and experienced improved survival. Adenosine receptors influence blood pressure and survival in salt-sensitive rats, and the impact of deleting adenosine receptors on blood pressure and survival depends on salt diet and sex.

8-Aminoguanosine and 8-Aminoguanine Exert Diuretic, Natriuretic, Glucosuric, and Antihypertensive Activity.

In vivo, guanine moieties in DNA, RNA, guanine nucleotides, or guanosine or guanine per se can undergo nitration (for example, by peroxynitrite) or hydroxylation (for example, by superoxide anion) on position 8 of the purine ring. Subsequent catabolism of these modified biomolecules leads to the production of a diverse group of 8-nitro, 8-amino, and 8-hydroxy guanosine and guanine compounds. Indeed, studies suggest the in vivo existence of 8-nitroguanosine, 8-nitroguanine, 8-aminoguanosine, 8-aminoguanine, 8-hydroxyguanosine, 8-hydroxy-2'-deoxyguanosine, and 8-hydroxyguanine. Since a multitude of these compounds exist in vivo, and since the renal effects of 8-substituted guanosine and guanine compounds are entirely unknown, we examined the effects of guanosine, guanine, 8-nitroguanosine, 8-nitroguanine, 8-hydroxyguanosine, 8-hydroxyguanine, 8-hydroxy-2'-deoxyguanosine, 8-aminoguanosine, and 8-aminoguanine (33.5 µmol/kg/min; intravenous infusion for 115 minutes) on excretion of sodium, potassium, and glucose in rats. Guanosine, 8-nitroguanosine, and 8-hydroxy-2'-deoxyguanosine had minimal natriuretic activity. Guanine, 8-nitroguanine, 8-hydroxyguanosine, and 8-hydroxyguanine had moderate natriuretic activity (increased sodium excretion by 9.4-, 7.8-, 7.1-, and 8.6-fold, respectively). In comparison with all other compounds, 8-aminoguanosine and 8-aminoguanine were highly efficacious and increased sodium excretion by 26.6- and 17.2-fold, respectively, exceeding that of a matched dose of amiloride (13.6-fold increase). 8-Aminoguanosine and 8-aminoguanine also increased glucose excretion by 12.1- and 12.2-fold, respectively, and decreased potassium excretion by 69.1 and 71.0%, respectively. Long-term radiotelemetry studies demonstrated that oral 8-aminoguanosine and 8-aminoguanine (5 mg/kg/day) suppressed deoxycorticosterone/salt-induced hypertension. These experiments demonstrate that some naturally occurring 8-substitued guanosine and guanine compounds, particularly 8-aminoguanosine and 8-aminoguanine, are potent and efficacious potassium-sparing diuretics/natriuretics that may represent a novel class of antihypertensive diuretics.

2-Methoxyestradiol, an endogenous 17ß-estradiol metabolite, inhibits microglial proliferation and activation via an estrogen receptor-independent mechanism.

17ß-Estradiol (estradiol) inhibits microglia proliferation. 2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with little affinity for estrogen receptors (ERs). We hypothesize that 2-ME inhibits microglial proliferation and activation and contributes to estradiol's inhibitory effects on microglia. We compared the effects of estradiol, 2-hydroxyestradiol [2-OE; estradiol metabolite produced by cytochrome P450 (CYP450)], and 2-ME [formed by catechol-O-methyltransferase (COMT) acting upon 2-OE] on microglial (BV2 cells) DNA synthesis, cell proliferation, activation, and phagocytosis. 2-ME and 2-OE were approximately three- and 10-fold, respectively, more potent than estradiol in inhibiting microglia DNA synthesis. The antimitogenic effects of estradiol were reduced by pharmacological inhibitors of CYP450 and COMT. Inhibition of COMT blocked the conversion of 2-OE to 2-ME and the antimitogenic effects of 2-OE but not 2-ME. Microglia expressed ERß and GPR30 but not ERα. 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERß agonist), but not 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (ERα agonist) or G1 (GPR30 agonist), inhibited microglial proliferation. The antiproliferative effects of estradiol, but not 2-OE or 2-ME, were partially reversed by ICI-182,780 (ERα/ß antagonist) but not by 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (ERα antagonist) or G15 (GPR30 antagonist). Lipopolysaccharide increased microglia iNOS and COX-2 expression and phagocytosing activity of microglia; these effects were inhibited by 2-ME. We conclude that in microglia, 2-ME inhibits proliferation, proinflammatory responses, and phagocytosis. 2-ME partially mediates the effects of estradiol via ER-independent mechanisms involving sequential metabolism of estradiol to 2-OE and 2-ME. 2-ME could be of potential therapeutic use in postischemic stroke injuries. Interindividual differences in estradiol metabolism might affect the individual's ability to recover from stroke.

NPY1-36 and PYY1-36 activate cardiac fibroblasts: an effect enhanced by genetic hypertension and inhibition of dipeptidyl peptidase 4.

Cardiac sympathetic nerves release neuropeptide Y (NPY)1-36, and peptide YY (PYY)1-36 is a circulating peptide; therefore, these PP-fold peptides could affect cardiac fibroblasts (CFs). We examined the effects of NPY1-36 and PYY1-36 on the proliferation of and collagen production ([(3)H]proline incorporation) by CFs isolated from Wistar-Kyoto (WKY) normotensive rats and spontaneously hypertensive rats (SHRs). Experiments were performed with and without sitagliptin, an inhibitor of dipeptidyl peptidase 4 [DPP4; an ectoenzyme that metabolizes NPY1-36 and PYY1-36 (Y1 receptor agonists) to NPY3-36 and PYY3-36 (inactive at Y1 receptors), respectively]. NPY1-36 and PYY1-36, but not NPY3-36 or PYY3-36, stimulated proliferation of CFs, and these effects were more potent than ANG II, enhanced by sitagliptin, blocked by BIBP3226 (Y1 receptor antagonist), and greater in SHR CFs. SHR CF membranes expressed more receptor for activated C kinase (RACK)1 [which scaffolds the Gi/phospholipase C (PLC)/PKC pathway] compared with WKY CF membranes. RACK1 knockdown (short hairpin RNA) and inhibition of Gi (pertussis toxin), PLC (U73122), and PKC (GF109203X) blocked the proliferative effects of NPY1-36. NPY1-36 and PYY1-36 stimulated collagen production more potently than did ANG II, and this was enhanced by sitagliptin and greater in SHR CFs. In conclusion, 1) NPY1-36 and PYY1-36, via the Y1 receptor/Gi/PLC/PKC pathway, activate CFs, and this pathway is enhanced in SHR CFs due to increased localization of RACK1 in membranes; and 2) DPP4 inhibition enhances the effects of NPY1-36 and PYY1-36 on CFs, likely by inhibiting the metabolism of NPY1-36 and PYY1-36. The implications are that endogenous NPY1-36 and PYY1-36 could adversely affect cardiac structure/function by activating CFs, and this may be exacerbated in genetic hypertension and by DPP4 inhibitors.

Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D.

The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N(6)-cyclopentyladenosine, CGS21680, or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase-associated protein-2 (Skp2; promotes proteolysis of p27(Kip1)) and upregulated levels of p27(Kip1) (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27(Kip1), cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine's inhibitory effects on Skp2 and stimulatory effects on p27(Kip1) and rescued HCASMCs from 2-chloroadenosine-mediated inhibition. Knockdown of p27(Kip1) also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 µmol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27(Kip1), and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37 424±18 371 pixels; treated, 10 352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin that controls cell-cycle progression.

Critical Role for the Adenosine Pathway in Controlling Simian Immunodeficiency Virus-Related Immune Activation and Inflammation in Gut Mucosal Tissues.

UNLABELLED: The role of the adenosine (ADO) pathway in human immunodeficiency virus type 1/simian immunodeficiency virus (HIV-1/SIV) infection remains unclear. We compared SIVsab-induced changes of markers related to ADO production (CD39 and CD73) and breakdown (CD26 and adenosine deaminase) on T cells from blood, lymph nodes, and intestine collected from pigtailed macaques (PTMs) and African green monkeys (AGMs) that experience different SIVsab infection outcomes. We also measured ADO and inosine (INO) levels in tissues by mass spectrometry. Finally, we assessed the suppressive effect of ADO on proinflammatory cytokine production after T cell receptor stimulation. The baseline level of both CD39 and CD73 coexpression on regulatory T cells and ADO levels were higher in AGMs than in PTMs. Conversely, high INO levels associated with dramatic increases in CD26 expression and adenosine deaminase activity were observed in PTMs during chronic SIV infection. Immune activation and inflammation markers in the gut and periphery inversely correlated with ADO and directly correlated with INO. Ex vivo administration of ADO significantly suppressed proinflammatory cytokine production by T cells in both species. In conclusion, the opposite dynamics of ADO pathway-related markers and contrasting ADO/INO levels in species with divergent proinflammatory responses to SIV infection support a key role of ADO in controlling immune activation/inflammation in nonprogressive SIV infections. Changes in ADO levels predominately occurred in the gut, suggesting that the ADO pathway may be involved in sparing natural hosts of SIVs from developing SIV-related gut dysfunction. Focusing studies of the ADO pathway on mucosal sites of viral replication is warranted. IMPORTANCE: The mechanisms responsible for the severe gut dysfunction characteristic of progressive HIV and SIV infection in humans and macaques are not completely elucidated. We report that ADO may play a key role in controlling immune activation/inflammation in nonprogressive SIV infections by limiting SIV-related gut inflammation. Conversely, in progressive SIV infection, significant degradation of ADO occurs, possibly due to an early increase of ADO deaminase complexing protein 2 (CD26) and adenosine deaminase. Our study supports therapeutic interventions to offset alterations of this pathway during progressive HIV/SIV infections. These potential approaches to control chronic immune activation and inflammation during pathogenic SIV infection may prevent HIV disease progression.