RESUMO
Screening of compounds comprising 8-substituted guanine revealed that 8-aminoguanosine and 8-aminoguanine cause diuresis/natriuresis/glucosuria, yet decrease potassium excretion. Subsequent investigations demonstrated that 8-aminoguanosine's effects are mediated by its metabolite 8-aminoguanine. The mechanism by which 8-aminoguanine causes diuresis/natriuresis/glucosuria involves inhibition of PNPase (purine nucleoside phosphorylase), which increases renal interstitial inosine levels. Additional evidence suggests that inosine, via indirect or direct adenosine A2B receptor activation, increases renal medullary blood flow which enhances renal excretory function. Likely, 8-aminoguanine has pleiotropic actions that also alter renal excretory function. Indeed, the antikaliuretic effects of 8-aminoguanine are independent of PNPase inhibition. 8-Aminoguanine is an endogenous molecule; nitrosative stress leads to production of biomolecules containing 8-nitroguanine moieties. Degradation of these biomolecules releases 8-nitroguanosine and 8-nitro-2'-deoxyguanosine which are converted to 8-aminoguanine. Also, guanosine and guanine per se may contribute to 8-aminoguanine formation. 8-Aminoinosine, 8-aminohypoxanthine, and 8-aminoxanthine likewise induce diuresis/natriuresis/glucosuria, yet do not reduce potassium excretion. Thus, there are several pharmacologically active 8-aminopurines with nuanced effects on renal excretory function. Chronic treatment with 8-aminoguanine attenuates hypertension in deoxycorticosterone/salt rats, prevents strokes, and increases lifespan in Dahl salt-sensitive rats on a high salt diet and attenuates the metabolic syndrome in rats; 8-aminoguanosine retards progression of pulmonary hypertension in rats and anemia and organ damage in sickle cell mice. 8-Aminoguanine reverses age-associated lower urinary tract dysfunction and retinal degeneration. 8-Aminopurines represent a new class of agents (and potentially endogenous factors) that have beneficial effects on the cardiovascular system and kidneys and may turn back the clock in age-associated diseases.
Assuntos
Sistema Cardiovascular , Guanina , Ratos , Camundongos , Animais , Ratos Endogâmicos Dahl , Guanina/metabolismo , Guanina/farmacologia , Natriurese , Sistema Cardiovascular/metabolismo , Potássio , Inosina/farmacologiaRESUMO
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.
Assuntos
Adenilil Ciclases , Diurese , Ratos , Humanos , Animais , Adenilil Ciclases/farmacologia , Células HEK293 , Diuréticos/farmacologia , Natriurese , Receptores Purinérgicos P1 , Inosina/farmacologiaRESUMO
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.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Dipiridamol/farmacologia , Infecções por HIV/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Adenosina/metabolismo , Biópsia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Estudos Cross-Over , Feminino , Citometria de Fluxo , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-IdadeRESUMO
Here, we tested the hypothesis that TNAP (tissue nonspecific alkaline phosphatase) modulates vascular responsiveness to norepinephrine. In the isolated, Tyrode's-perfused rat mesentery, 50 µmol/L of L-p-bromotetramisole (L-p-BT; selective TNAP inhibitor, Ki=56 µmol/L) significantly reduced TNAP activity and caused a significant 9.0-fold rightward-shift in the norepinephrine concentration versus vasoconstriction relationship. At 100 µmol/L, L-p-BT further reduced mesenteric TNAP activity and caused an additional significant right-shift of the norepinephrine concentration versus vasoconstriction relationship. A higher concentration (200 µmol/L) of L-p-BT had no further effect on either mesenteric TNAP activity or norepinephrine-induced vasoconstriction. L-p-BT did not alter vascular responses to vasopressin, thus ruling-out nonspecific suppression of vascular reactivity. Since in the rat mesenteric vasculature α1-adrenoceptors mediate norepinephrine-induced vasoconstriction, these finding indicate that TNAP inhibition selectively interferes with α1-adrenoceptor signaling. Additional experiments showed that the effects of TNAP inhibition on norepinephrine-induced vasoconstriction were not mediated by accumulation of pyrophosphate or ATP (TNAP substrates) nor by reduced adenosine levels (TNAP product). TNAP inhibition significantly reduced the Hillslope of the norepinephrine concentration versus vasoconstriction relationship from 1.8±0.2 (consistent with positive cooperativity of α1-adrenoceptor signaling) to 1.0±0.1 (no cooperativity). Selective activation of A1-adenosine receptors, which are known to participate in coincident signaling with α1-adrenoceptors, reversed the suppressive effects of L-p-BT on norepinephrine-induced vasoconstriction. In vivo, L-p-BT administration achieved plasma levels of ≈60 µmol/L and inhibited mesenteric vascular responses to exogenous norepinephrine and sympathetic nerve stimulation. TNAP modulates vascular responses to norepinephrine likely by affecting positive cooperativity of α1-adrenoceptor signaling via a mechanism involving A1 receptor signaling.
Assuntos
Fosfatase Alcalina/metabolismo , Proteínas de Membrana/metabolismo , Mesentério/efeitos dos fármacos , Norepinefrina/farmacologia , Tetramizol/análogos & derivados , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Antagonistas do Receptor A1 de Adenosina/farmacologia , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/genética , Animais , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Mesentério/metabolismo , Ratos , Tetramizol/farmacologia , Xantinas/farmacologiaRESUMO
Estradiol may antagonize the adverse cardiovascular effects of angiotensin II (Ang II). We investigated the effects of 2-methoxyestradiol (2-ME), a nonestrogenic estradiol metabolite, on Ang II-induced cardiovascular and renal injury in male rats. First, we determined the effects of 2-ME on Ang II-induced acute changes in blood pressure, renal hemodynamics, and excretory function. Next, we investigated the effects of 2-ME and 2-hydroxyestardiol (2-HE) on hypertension and cardiovascular and renal injury induced by chronic infusion of Ang II. Furthermore, the effects of 2-ME on blood pressure and cardiovascular remodeling in the constricted aorta (CA) rat model and on isoproterenol-induced (ISO) cardiac hypertrophy and fibrosis were examined. 2-ME had no effects on Ang II-induced acute changes in blood pressure, renal hemodynamics, or glomerular filtration rate. Both 2-ME and 2-HE reduced hypertension, cardiac hypertrophy, proteinuria, and mesangial expansion induced by chronic Ang II infusions. In CA rats, 2-ME attenuated cardiac hypertrophy and fibrosis and reduced elevated blood pressure above the constriction. Notably, 2-ME reduced both pressure-dependent (above constriction) and pressure-independent (below constriction) vascular remodeling. 2-ME had no effects on ISO-induced renin release yet reduced ISO-induced cardiac hypertrophy and fibrosis. This study shows that 2-ME protects against cardiovascular and renal injury due to chronic activation of the renin-angiotensin system. This study reports for the first time that in vivo 2-ME reduces trophic (pressure-independent) effects of Ang II and related cardiac and vascular remodeling.
Assuntos
2-Metoxiestradiol/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/prevenção & controle , Hipertrofia Ventricular Esquerda/prevenção & controle , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Remodelação Vascular/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Angiotensina II , Animais , Fibrose , Taxa de Filtração Glomerular/efeitos dos fármacos , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/induzido quimicamente , Hipertrofia Ventricular Esquerda/fisiopatologia , Isoproterenol , Rim/patologia , Rim/fisiopatologia , Nefropatias/induzido quimicamente , Nefropatias/fisiopatologia , Masculino , Ratos Sprague-Dawley , Sistema Renina-Angiotensina/efeitos dos fármacosRESUMO
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.
Assuntos
Proliferação de Células , Quimiocina CXCL12/metabolismo , Fibroblastos , Hipertensão/metabolismo , Insulisina , Miocárdio/patologia , Receptores CXCR4 , Animais , Pressão Sanguínea/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Insulisina/antagonistas & inibidores , Insulisina/metabolismo , Neuropeptídeo Y/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismo , Transdução de Sinais , Fosfato de Sitagliptina/farmacologia , Ubiquitina/metabolismoRESUMO
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.
Assuntos
Pressão Sanguínea/fisiologia , Regulação da Expressão Gênica , RNA/genética , Receptores Purinérgicos P1/genética , Cloreto de Sódio na Dieta/farmacologia , Animais , Dieta Hipossódica , Modelos Animais de Doenças , Feminino , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos Dahl , Receptores Purinérgicos P1/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Activated cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), and glomerular mesangial cells (GMCs) proliferate, cause hypertrophy, and produce collagen; in this way, activated CFs contribute to cardiac fibrosis, and activated PGVSMCs and GMCs promote renal fibrosis. In heart and kidney diseases, SDF-1α (stromal cell-derived factor 1α; endogenous CXCR4 [C-X-C motif chemokine receptor 4] receptor agonist) levels are often elevated; therefore, it is important to know whether and how the SDF-1α/CXCR4 axis activates CFs, PGVSMCs, or GMCs. METHODS AND RESULTS: Here we investigated whether SDF-1α activates CFs, PGVSMCs, and GMCs to proliferate, hypertrophy, or produce collagen. DPP4 (dipeptidyl peptidase 4) inactivates SDF-1α and previous experiments show that growth-promoting peptides have greater effects in cells from genetically-hypertensive animals. Therefore, we performed experiments in the absence and presence of sitagliptin (DPP4 inhibitor) and in cells from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats. Our studies show (1) that spontaneously hypertensive and Wistar-Kyoto rat CFs, PGVSMCs, and GMCs express CXCR4 receptors and DPP4 activity; (2) that chronic treatment with physiologically relevant concentrations of SDF-1α causes concentration-dependent increases in the proliferation (cell number) and hypertrophy (3H-leucine incorporation) of and collagen production (3H-proline incorporation) by CFs, PGVSMCs, and GMCs; (3) that sitagliptin augments these effects of SDF-1α; (4) that interactions between SDF-1α and sitagliptin are greater in spontaneously hypertensive rat cells; (5) that CXCR4 antagonism (AMD3100) blocks all effects of SDF-1α; and (6) that SDF-1α/CXCR4 signal transduction likely involves the RACK1 (receptor for activated C kinase 1)/Gßγ/PLC (phospholipase C)/PKC (protein kinase C) signaling complex. CONCLUSIONS: The SDF-1α/CXCR4 axis drives proliferation and hypertrophy of and collagen production by CFs, PGVSMCs, and GMCs, particularly in cells from genetically hypertensive animals and when DPP4 is inhibited.
Assuntos
Cardiomiopatia Hipertrófica/genética , Quimiocina CXCL12/genética , Colágeno/biossíntese , Regulação da Expressão Gênica , Células Mesangiais/patologia , Músculo Liso Vascular/patologia , Miocárdio/patologia , Animais , Western Blotting , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Proliferação de Células , Células Cultivadas , Quimiocina CXCL12/biossíntese , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Células Mesangiais/metabolismo , Microvasos/metabolismo , Microvasos/patologia , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , RNA/genética , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR4/biossíntese , Receptores CXCR4/genética , Artéria Renal/metabolismo , Artéria Renal/patologiaRESUMO
Tissue nonspecific alkaline phosphatase (TNAP) contributes to the production of adenosine by the kidney, and A1-receptor activation enhances renovascular responses to norepinephrine. Therefore, we hypothesized that TNAP regulates renovascular responsiveness to norepinephrine. In isolated, perfused rat kidneys, the TNAP inhibitor l-p-bromotetramisole (0.1 mmol/L) decreased renal venous levels of 5'-AMP (adenosine precursor) and adenosine by 61% (P<0.0384) and 62% (P=0.0013), respectively, at 1 hour into treatment and caused a 10-fold rightward shift of the concentration-response relationship to exogenous norepinephrine (P<0.0001). Similarly, 2 other TNAP inhibitors, levamisole (1 mmol/L) and 2,5-dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (0.02 mmol/L), also right shifted the concentration-response relationship to norepinephrine. The ability of TNAP inhibition to blunt renovascular responses to norepinephrine was mostly prevented or reversed by restoring A1-adenosinergic tone with the A1-receptor agonist 2-chloro-N6-cyclopentyladenosine (100 nmol/L). All 3 TNAP inhibitors also attenuated renovascular responses to renal sympathetic nerve stimulation, suggesting that TNAP inhibition attenuates renovascular responses to endogenous norepinephrine. In control propranolol-pretreated rats, acute infusions of norepinephrine (10 µg/kg/min) increased mean arterial blood pressure from 95±5 mm Hg to a peak of 169±4 mm Hg and renovascular resistance from 12±2 mm Hg/mL/min to a peak of 55±12 mm Hg/mL/min; however, in rats also treated with intravenous l-p-bromotetramisole (30 mg/kg), the pressor and renovascular effects of norepinephrine were significantly attenuated (blood pressure: basal and peak, 93±7 and 146±6 mm Hg, respectively; renovascular resistance: basal and peak, 13±2 and 29±5 mm Hg/mL/min, respectively). TNAP inhibitors attenuate renovascular and blood pressure responses to norepinephrine, suggesting that TNAP participates in the regulation of renal function and blood pressure.