Zaichuan Mi

Exogenous and Endogenous Adenosine Inhibits Fetal Calf Serum–Induced Growth of Rat Cardiac Fibroblasts Role of A2B Receptors

BACKGROUND Because proliferation of cardiac fibroblasts participates in cardiac hypertrophy/remodeling associated with hypertension and myocardial infarction, it is important to elucidate factors regulating cardiac fibroblast proliferation. Adenosine, a nucleoside abundantly produced by cardiac cells, is antimitogenic vis-à-vis vascular smooth muscle cells; however, the effect of adenosine on cardiac fibroblast proliferation is unknown. The objective of this study was to characterize the effects of exogenous and endogenous (cardiac fibroblast-derived) adenosine on cardiac fibroblast proliferation. METHODS AND RESULTS Growth-arrested cardiac fibroblasts were stimulated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), or modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; adenosine deaminase inhibitor); (2) dipyridamole (adenosine transport blocker); and (3) iodotubericidin (adenosine kinase inhibitor). All of these agents inhibited, in a concentration-dependent manner, FCS-induced cardiac fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation) and cell counting. EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 2.3- to 5.6-fold when added separately to cardiac fibroblasts, and EHNA+iodotubericidin or EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by >690-fold. Both KF17837 (selective A2 antagonist) and DPSPX (nonselective A2 antagonist) but not DPCPX (selective A1 antagonist) blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA synthesis, suggesting the involvement of A2A and/or A2B but excluding the participation of A1 receptors. The lack of effect of CGS21680 (selective A2A agonist) excluded involvement of A2A receptors and suggested a major role for A2B receptors. This conclusion was confirmed by the rank order potencies of four adenosine analogues. CONCLUSIONS Cardiac fibroblasts synthesize adenosine, and exogenous and cardiac fibroblast-derived adenosine inhibits cardiac fibroblast proliferation via activation of A2B receptors. Cardiac fibroblast-derived adenosine may regulate cardiac hypertrophy and/or remodeling by modulating cardiac fibroblast proliferation.

Adenosine Inhibits Growth of Human Aortic Smooth Muscle Cells Via A2B Receptors

Adenosine inhibits rat vascular smooth muscle cell (SMC) growth. However, the effects of adenosine on human vascular SMC proliferation and synthesis of extracellular matrix proteins, such as collagen, are unknown. The objective of this study was to characterize the effects of exogenous and endogenous (SMC-derived) adenosine on human aortic SMC proliferation and collagen synthesis. Growth-arrested SMCs were stimulated with 2.5% fetal calf serum (FCS) in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), and with agents that increase endogenous adenosine levels, including erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), dipyridamole, and iodotubericidin. All of these agents inhibited in a concentration-dependent manner FCS-induced SMC proliferation as assessed by DNA synthesis (3H-thymidine incorporation) and cell counting, as well as collagen synthesis (3H-proline incorporation). EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 1.7-fold to 18-fold when added separately to SMCs, and EHNA+iodotubericidin and EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by more than 392-fold. Both KF17837 (selective A2 antagonist) and DPSPX (A1/A2 antagonist), but not DPCPX (selective A1 antagonist), blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA and collagen synthesis, suggesting the involvement of A2A and/or A2B, but excluding the participation of A1, receptors. The lack of effect of CGS21680 (selective A2A agonist), excluded involvement of A2A receptors and suggested a major role for A2B receptors. A comparison of the inhibitory potencies of 2-chloroadenosine, N6-cyclopentyladenosine (selective A1 agonist), NECA (A1/A2 agonist), and MECA (A1/A2 agonist) were consistent with an A2B receptor subtype mediating the inhibitory effects of adenosine on human aortic SMC proliferation. In conclusion, human aortic SMCs synthesize adenosine, and exogenous as well as endogenous (SMC-derived) adenosine inhibits SMC proliferation and collagen synthesis via activation of A2B receptors.

A2B Receptors Mediate the Antimitogenic Effects of Adenosine in Cardiac Fibroblasts

Adenosine inhibits growth of cardiac fibroblasts; however, the adenosine receptor subtype that mediates this antimitogenic effect remains undefined. Therefore, the goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat left ventricular cardiac fibroblasts, PDGF-BB (25 ng/mL) stimulated DNA synthesis (3H-thymidine incorporation), cellular proliferation (cell number), collagen synthesis (3H-proline incorporation), and MAP kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5′-N-methylcarboxamidoadenosine, but not N6-cyclopentyladenosine, 4-aminobenzyl-5′-N-methylcarboxamidoadenosine, or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A2B receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1,3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5′-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A2 receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A2B receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and collagen synthesis. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5′-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A2B receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of CF growth by activating A2B receptors coupled to inhibition of MAP kinase activity. Thus, A2B receptors may play a critical role in regulating cardiac remodeling associated with CF proliferation. Pharmacologic or molecular biological activation of A2B receptors may prevent cardiac remodeling associated with hypertension, myocardial infarction, and myocardial reperfusion injury after ischemia.

Cerebrospinal fluid adenosine concentration and uncoupling of cerebral blood flow and oxidative metabolism after severe head injury in humans.

OBJECTIVE Uncoupling of cerebral blood flow (CBF) and oxidative metabolism is observed after severe head injury in comatose patients; however, the mechanism(s) involved remain undefined. Adenosine can produce cerebral vasodilation and reduce neuronal activity and is a possible mediator of uncoupling. We hypothesized that cerebrospinal fluid (CSF) adenosine concentrations would be increased during uncoupling of CBF and oxidative metabolism, defined as a narrow arterio-jugular venous oxygen difference [D(a-v)O2 4 vol%] after head injury. METHODS Adenosine concentrations were measured using fluorescent-based high-pressure liquid chromatography in 67 CSF samples obtained from 13 comatose (Glasgow Coma Scale score 7) adult patients who sustained a severe closed head injury. At the time each sample was obtained, CBF was measured by the xenon-133 method, and blood samples were obtained for determination of D(a-v)O2. RESULTS CSF adenosine concentration was negatively associated with D(a-v)O2 (P < 0.05, generalized multivariate linear regression model). In addition, CSF adenosine concentration was increased when D(a-v)O2 was 4 versus > 4 vol% (38.5 [3.2-306.3] versus 14.0 [2.7-795.5] nmol/L, respectively, median [range]; P < 0.025) and in patients who died versus survivors (40.1 [6.9-306.3] versus 12.9 [2.7-795.5] nmol/L, respectively, median [range]; P < 0.001). CONCLUSION The association between increased CSF adenosine concentration and a reduction in global cross-brain extraction of oxygen supports a regulatory role for adenosine in the complex balance between CBF and oxidative and nonoxidative metabolism severe head injury in humans.

Smooth Muscle Cell–Derived Adenosine Inhibits Cell Growth

Several endogenous factors generated within the vessel wall have been implicated in contributing to the vascular remodeling process associated with hypertension and atherosclerosis. Furthermore, substances generated by smooth muscle cells (SMCs) are known to regulate SMC proliferation in an autocrine fashion. Adenosine is a vasodilator synthesized by SMCs, and exogenous adenosine inhibits SMC proliferation. However, whether adenosine produced endogenously has antimitogenic effects is not known. Hence, we evaluated the effects of SMC-derived adenosine on 2.5% fetal calf serum-induced proliferation of rat aortic SMCs. SMC proliferation was assayed by measurement of DNA synthesis ([3H]thymidine incorporation) and cell counting. To determine the effects of endogenous adenosine on SMC proliferation, we stimulated growth-arrested SMCs with 2.5% fetal calf serum in the presence and absence of modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; inhibits adenosine deaminase), (2) dipyridamole (blocks adenosine transport and inhibits phosphodiesterase), (3) dipyridamole plus EHNA, and (4) adenosine with or without EHNA. [3H]Thymidine incorporation and cell number were measured after 24 and 96 hours, respectively. EHNA and dipyridamole inhibited both FCS-induced DNA synthesis and cell proliferation in a concentration-dependent manner. Furthermore, extracellular (in medium) adenosine levels were significantly increased when cultured cells were treated with EHNA, and the inhibitory effects of dipyridamole as well as exogenous adenosine were enhanced in the presence of EHNA. Additionally, the inhibitory effects of dipyridamole and EHNA on DNA synthesis were significantly reduced in the presence of KF17837, an A2 adenosine receptor antagonist. These results indicate that SMC-derived adenosine can inhibit SMC proliferation. Hence, it is possible that a defect in localized adenosine synthesis within the vessel wall could contribute to vascular thickening and neointima formation.

Extracellular 2′,3′-cAMP Is a Source of Adenosine

We discovered that renal injury releases 2′,3′-cAMP (positional isomer of 3′,5′-cAMP) into the interstitium. This finding motivated a novel hypothesis: renal injury leads to activation of an extracellular 2′,3′-cAMP-adenosine pathway (i.e. metabolism of extracellular 2′,3′-cAMP to 3′-AMP and 2′-AMP, which are metabolized to adenosine, a retaliatory metabolite). In isolated rat kidneys, arterial infusions of 2′,3′-cAMP (30 μmol/liter) increased the mean venous secretion of 3′-AMP (3,400-fold), 2′-AMP (26,000-fold), adenosine (53-fold), and inosine (adenosine metabolite, 30-fold). Renal injury with metabolic inhibitors increased the mean secretion of 2′,3′-cAMP (29-fold), 3′-AMP (16-fold), 2′-AMP (10-fold), adenosine (4.2-fold), and inosine (6.1-fold) while slightly increasing 5′-AMP (2.4-fold). Arterial infusions of 2′-AMP and 3′-AMP increased secretion of adenosine and inosine similar to that achieved by 5′-AMP. Renal artery infusions of 2′,3′-cAMP in vivo increased urinary excretion of 2′-AMP, 3′-AMP and adenosine, and infusions of 2′-AMP and 3′-AMP increased urinary excretion of adenosine as efficiently as 5′-AMP. The implications are that 1) in intact organs, 2′-AMP and 3′-AMP are converted to adenosine as efficiently as 5′-AMP (previously considered the most important adenosine precursor) and 2) because 2′,3′-cAMP opens mitochondrial permeability transition pores, a pro-apoptotic/pro-necrotic process, conversion of 2′,3′-cAMP to adenosine by the extracellular 2′,3′-cAMP-adenosine pathway would protect tissues by reducing a pro-death factor (2′,3′-cAMP) while increasing a retaliatory metabolite (adenosine).

Effects of dipeptidyl peptidase iv inhibition on arterial blood pressure.

1 The aim of the present study was to determine whether inhibition of dipeptidyl peptidase IV (DPP IV) elevates arterial blood pressure and whether any such effect is dependent on genetic background, the sympathetic nervous system and Y1 receptors. The rationale behind this study was that: (i) neuropeptide (NP) Y1–36 and peptide YY1–36 (PYY1–36) are endogenous Y1 receptor agonists and are metabolised by DPP IV to NPY3–36 and PYY3–36, which are not Y1 but rather selective Y2 receptor agonists; (ii) Y1 receptors mediate vasoconstriction, whereas Y2 receptors have little effect on vascular tone; (iii) vaso‐constrictor effect of the Y1 receptor is enhanced in spontaneously hypertensive rats (SHR) compared with normotenisve Wistar‐Kyoto (WKY) rats; and (iv) NPY1–36 is released from sympathetic nerve terminals. 2 We examined the effects of acute administration of 3‐N‐[(2S,3S)‐2‐amino‐3‐methylpentanoyl]‐1,3‐thiazolidine (P32/98; a DPP IV inhibitor) on arterial blood pressure in anaesthetized adult SHR and WKY rats in the absence and presence of either captopril, hydralazine or chlorisondamine to lower basal mean arterial blood pressure (MABP) by different mechanisms (inhibition of angiotensin‐converting enzyme, direct vasodilation and ganglionic blockade, respectively). 3 In naïve SHR with severely elevated basal blood pressures (MABP = 176 ± 3 mmHg; n = 4), i.v. boluses (1, 3 and 10 mg/kg) of P32/98 did not affect blood pressure. 4 When basal blood pressure was reduced by pretreatment of SHR with either captopril (30 mg/kg, i.v.; MABP = 116 ± 3 mmHg; n = 9) or hydralazine (5 mg/kg, i.p.; MABP = 84 ± 3 mmHg; n = 7), P32/98 (1, 3 and 10 mg/kg) caused significant dose‐related increases in arterial blood pressure (4 ± 2, 10 ± 2 and 12 ± 3 mmHg in the captopril‐pretreated group, respectively (P < 0.01); 5 ± 2, 8 ± 3 and 11 ± 4 mmHg in the hydralazine‐pretreated group, respectively (P < 0.01)). 5 The increases in arterial blood pressure induced by P32/98 in captopril‐ or hydralazine‐pretreated SHR were entirely blocked by pretreatment with the selective Y1 receptor antagonist N2‐(diphenylacetyl)‐N‐[(4‐hydroxyphenyl)methyl]‐d‐arginine amide (BIBP 3226; 6 mg/kg per h). 6 When basal blood pressure was reduced in SHR by pretreatment with chlorisondamine (10 mg/kg, s.c.; MABP = 108 ± 4 mmHg; n = 7), inhibition of DPP IV with P32/98 did not affect arterial blood pressure. Basal heart rate in chlorisondamine‐treated SHR was significantly reduced compared with naïve SHR, captopril‐pretreated SHR and hydralazine‐pretreated SHR, indicating effectiveness of ganglionic blockade. 7 Unlike the results in genetically hypertensive animals, in normotensive WKY rats pretreated with captopril (30 mg/kg, i.v.; MABP = 81 ± 4 mmHg; n = 6), or hydralazine (5 mg/kg, i.p.; MABP = 63 ± 4 mmHg; n = 4) or chlorisondamine (10 mg/kg, s.c.; MABP = 63 ± 4 mmHg; n = 5), P32/98 did not affect arterial blood pressure. 8 We conclude that, in genetically susceptible animals, inhibition of DPP IV increases arterial blood pressure via Y1 receptors when elevated blood pressure is reduced with antihypertensive drugs provided that the sympathetic nervous system is functional. The results suggest vigilance because DPP IV inhibitors are used more widely in hypertensive patients treated with antihypertensive drugs.

Increased adenosine in cerebrospinal fluid after severe traumatic brain injury in infants and children: association with severity of injury and excitotoxicity.

Objectives To measure adenosine concentration in the cerebrospinal fluid of infants and children after severe traumatic brain injury and to evaluate the contribution of patient age, Glasgow Coma Scale score, mechanism of injury, Glasgow Outcome Score, and time after injury to cerebrospinal fluid adenosine concentrations. To evaluate the relationship between cerebrospinal fluid adenosine and glutamate concentrations in this population. Design Prospective survey. Setting Pediatric intensive care unit in a university-based children’s hospital. Patients Twenty-seven critically ill infants and children who had severe traumatic brain injury (Glasgow Coma Scale <8), who required placement of an intraventricular catheter and drainage of cerebrospinal fluid as part of their neurointensive care. Interventions None. Measurements and Main Results Patients ranged in age from 2 months to 14 yrs. Cerebrospinal fluid samples (n = 304) were collected from 27 patients during the first 7 days after traumatic brain injury. Control cerebrospinal fluid samples were obtained from lumbar puncture on 21 infants and children without traumatic brain injury or meningitis. Adenosine concentration was measured by using high-pressure liquid chromatography. Adenosine concentration was increased markedly in cerebrospinal fluid of children after traumatic brain injury vs. controls (p < .001). The increase in cerebrospinal fluid adenosine was independently associated with Glasgow Coma Scale ≤4 vs. >4 and time after injury (both p < .005). Cerebrospinal fluid adenosine concentration was not independently associated with either age (≤4 vs. >4 yrs), mechanism of injury (abuse vs. other), or Glasgow Outcome Score (good/moderately disabled vs. severely disabled, vegetative, or dead). Of the 27 patients studied, 18 had cerebrospinal fluid glutamate concentration previously quantified by high-pressure liquid chromatography. There was a strong association between increases in cerebrospinal fluid adenosine and glutamate concentrations (p < .005) after injury. Conclusions Cerebrospinal fluid adenosine concentration is increased in a time- and severity-dependent manner in infants and children after severe head injury. The association between cerebrospinal fluid adenosine and glutamate concentrations may reflect an endogenous attempt at neuroprotection against excitotoxicity after severe traumatic brain injury.

Interstitial brain adenosine and xanthine increase during jugular venous oxygen desaturations in humans after traumatic brain injury.

ObjectiveAdenosine decreases the cerebral metabolic rate for oxygen and increases cerebral blood flow, and it may play an important role in cerebrometabolic and cerebrovascular responses to hypoperfusion after traumatic brain injury. Jugular venous oxygen saturation is monitored after traumatic brain injury to assess brain oxygen extraction, and desaturations may reflect secondary brain insults. We hypothesized that brain interstitial adenosine and related purine metabolites would be increased during jugular venous oxygen saturation desaturations (<50%) and determined associations between the purines, lactate, and glucose to assess the role of adenosine during secondary insults in humans. DesignStudy of critically ill adults with severe traumatic brain injury. SettingAdult neurointensive care unit. PatientsWe prospectively defined periods of normal saturation and desaturation in six patients after severe traumatic brain injury. InterventionsDuring these periods, cerebral microdialysis samples of brain interstitial fluid were collected, and adenosine and purine metabolites were measured by high-pressure liquid chromatography. Measurements and Main Results Adenosine increased 3.1-fold and xanthine increased 2.5-fold during desaturation periods (both p < .05 vs. normal saturation period, signed rank). Adenosine, xanthine, hypoxanthine, and cyclic-adenosine monophosphate correlated with lactate over both study periods (r2 = .32, .14, .31, .07, and .26, respectively, all p < .05, Pearson product moment correlation). ConclusionThe marked increases in interstitial brain adenosine that occur during jugular venous oxygen desaturations suggest that adenosine may play an important role during periods of secondary insults after traumatic brain injury. The correlation of these metabolites with lactate further suggests that adenosine is increased during periods of enhanced glycolytic metabolism.

Cardiac Fibroblasts Express the cAMP-Adenosine Pathway

The extracellular “cAMP-adenosine pathway” refers to the local production of adenosine mediated by cAMP egress into the extracellular space, conversion of cAMP to AMP by ectophosphodiesterase, and the metabolism of AMP to adenosine by ecto-5′-nucleotidase. The goal of this study was to assess whether the cAMP-adenosine pathway limits cardiac fibroblast growth. Studies were conducted in ventricular cardiac fibroblasts maintained in 3-dimensional cultures. Addition of exogenous cAMP to cardiac fibroblasts increased extracellular levels of AMP, adenosine, and inosine in a concentration-dependent and time-dependent manner. This effect was attenuated by blockade of total phosphodiesterase activity (3-isobutyl-1-methylxanthine), ectophosphodiesterase activity (high concentration of 1,3-dipropyl-8-p-sulfophenylxanthine), or ecto-5′-nucleotidase (&agr;, &bgr;-methylene-adenosine-5′-diphosphate). Treatment with exogenous cAMP inhibited cell growth as assessed by DNA synthesis (3H-thymidine incorporation), cell proliferation (cell counts), and protein synthesis (3H-leucine incorporation). Antagonism of A2 (KF17837) or A1/A2 (low concentration of 1,3-dipropyl-8-p-sulfophenylxanthine), but not A1 (8-cyclopentyl-1,3-dipropylxanthine), adenosine receptors blocked the growth-inhibitory effects of exogenous cAMP, but not the growth inhibitory effects of 8-bromo-cAMP (stable cAMP analogue). The growth-inhibitory effects of exogenous cAMP were enhanced by the combined inhibition of adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl) adenine] and adenosine kinase (iodotubercidin). In conclusion, the extracellular cAMP-adenosine pathway exists in cardiac fibroblasts and attenuates cell growth. Pharmacological augmentation of this pathway could abate pathological cardiac remodeling in heart disease.