Implication of adenosine A2A receptors in hypotension-induced vasodilation and cerebral blood flow autoregulation in rat pial arteries

This study aimed to evaluate the role for adenosine A2A receptors in the autoregulatory vasodilation to hypotension in relation with cerebral blood flow (CBF) autoregulation in rat pial arteries. Changes in pial artery diameters were observed directly through a closed cranial window. Vasodilation induced by adenosine was markedly suppressed by ZM 241385 (1 micromol/l, A2A antagonist) and alloxazine (1 micromol/l, A2B antagonist), but not by 8-cyclopentyltheophylline (CPT, 1 micromol/l, A1 antagonist). CGS-21680-induced vasodilation was more strongly inhibited by ZM 241385 (25.3-fold; P<0.05) than by alloxazine. In contrast, 5'-N-ethylcarboxamido-adenosine (NECA)-induced vasodilation was more prominently suppressed by alloxazine (12.0-fold; P<0.001) than by ZM 241385. The autoregulatory vasodilation in response to acute hypotension of the pial arteries was significantly suppressed by ZM 241385, but not by CPT and alloxazine. Consistent with this finding, the lower limit of CBF autoregulation significantly shifted to a higher blood pressure by 1 micromol/l of ZM 241385 (53.0+/-3.9 mm Hg to 69.2+/-2.9 mm Hg, P<0.01) and 10 micromol/l of glibenclamide (54.7+/-6.5 mm Hg to 77.9+/-4.2 mm Hg, P<0.001), but not by CPT and alloxazine. Thus, it is suggested that adenosine-induced vasodilation of the rat pial artery is mediated via activation of adenosine A2A and A2B receptors, but not by A1 subtype, and activation of adenosine A2A receptor preferentially contributes to the autoregulatory vasodilation via activation of ATP-sensitive K+ channels in response to hypotension and maintenance of CBF autoregulation.     

Contribution of adenosine A2A and A2B receptors and heme oxygenase to AMPA-induced dilation of pial arterioles in rats

Nitric oxide (NO) has been implicated in mediation of cerebral vasodilation during neuronal activation and, specifically, in pharmacological activation of N-methyl-d-aspartate (NMDA) and kainate receptors. Possible mediators of cerebral vasodilation to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have not been well studied in mature brain, although heme oxygenase (HO) activity has been implicated in newborn pigs. In anesthetized rats, 5 min of topical superfusion of 30 and 100 microM AMPA on the cortical surface through a closed cranial window resulted in increases in pial arteriolar diameter. The dilatory response to AMPA was not inhibited by superfusion of an NO synthase inhibitor, a cyclooxygenase-2 inhibitor, or a cytochrome P-450 epoxygenase inhibitor, all of which have been shown to inhibit the cortical blood flow response to sensory activation. However, the 48 +/- 13% dilation to 100 microM AMPA was attenuated 56-71% by superfusion of the adenosine A(2A) receptor antagonist ZM-241385, the A(2B) receptor antagonist alloxazine, and the HO inhibitor chromium mesoporphyrin. Combination of the latter three inhibitors did not attenuate the dilator response more than the individual inhibitors, whereas an AMPA receptor antagonist fully blocked the vasodilation to AMPA. These results indicate that cortical pial arteriolar dilation to AMPA does not require activation of NO synthase, cyclooxygenase-2, or cytochrome P-450 epoxygenase but does depend on activation of adenosine A(2A) and A(2B) receptors. In addition, CO derived from HO appears to play a role in the vascular response to AMPA receptor activation in mature brain by a mechanism that is not additive with that of adenosine receptor activation.     

Adenosine receptors mediate glutamate-evoked arteriolar dilation in the rat cerebral cortex

We tested the hypothesis that adenosine (Ado) mediates glutamate-induced vasodilation in the cerebral cortex by monitoring pial arteriole diameter in chloralose-anesthetized rats equipped with closed cranial windows. Topical application of 100 microM glutamate and 100 microM N-methyl-d-aspartate (NMDA) dilated pial arterioles (baseline diameter 25 +/- 2 microm) by 17 +/- 1% and 18 +/- 4%, respectively. Coapplication of the nonselective Ado receptor antagonist theophylline (Theo; 10 microM) significantly reduced glutamate- and NMDA-induced vasodilation to 4 +/- 2% (P < 0.01) and 6 +/- 2% (P < 0.05), whereas the Ado A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM) had no effect. Moreover, application of the Ado A(2A) receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl)phenol (ZM-241385), either by superfusion (0.1 microM, 1 microM) or intravenously (1 mg/kg), significantly inhibited the pial arteriole dilation response to glutamate. Neither Theo nor ZM-241385 affected vascular reactivity to mild hypercapnia induced by 5% CO(2) inhalation. These results suggest that Ado contributes to the dilation of rat cerebral arterioles induced by exogenous glutamate, and that the Ado A(2A) receptor subtype may be involved in this dilation response.     

The A2a/A2b receptor antagonist ZM-241385 blocks the cardioprotective effect of adenosine agonist pretreatment in in vivo rat myocardium

There is increasing evidence for interactions among adenosine receptor subtypes in the brain and heart. The purpose of this study was to determine whether the adenosine A(2a) receptor modulates the infarct size-reducing effect of preischemic administration of adenosine receptor agonists in intact rat myocardium. Adult male rats were submitted to in vivo regional myocardial ischemia (25 min) and 2 h reperfusion. Vehicle-treated rats were compared with rats pretreated with the A(1) agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA, 10 mug/kg), the nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 mug/kg), or the A(2a) agonist 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-methylcarboxamidoadenosine (CGS-21680, 20 mug/kg). Additional CCPA- and NECA-treated rats were pretreated with the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 mug/kg), the A(2a)/A(2b) antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM-241385, 1.5 mg/kg) or the A(3) antagonist 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523, 2 mg/kg). CCPA and NECA reduced myocardial infarct size by 50% and 35%, respectively, versus vehicle, but CGS-21680 had no effect. DPCPX blunted the bradycardia associated with CCPA and NECA, whereas ZM-241385 attenuated their hypotensive effects. Both DPCPX and ZM-241385 blocked the protective effects of CCPA and NECA. The A(3) antagonist did not alter the hemodynamic effects of CCPA or NECA, nor did it alter adenosine agonist cardioprotection. None of the antagonists alone altered myocardial infarct size. These findings suggest that although preischemic administration of an A(2a) receptor agonist does not induce cardioprotection, antagonism of the A(2a) and/or the A(2b) receptor blocks the cardioprotection associated with adenosine agonist pretreatment.     

Mechanism of adenosine-induced vasodilation in rat diaphragm microcirculation

The mechanism of adenosine-induced vasodilation in rat diaphragm microcirculation was investigated using laser Doppler flowmetry. Adenosine (10(-5), 3.2 x 10(-5), and 10(-4) M), the nonselective adenosine agonist 5'-N-ethylcarboxamido-adenosine (NECA) (10(-8)-10(-7) M), the specific A(2A) agonist 2-p-(2-carboxyethyl)phenyl-amino-5'-N-ethyl carboxamidoadenosine (CGS-21680) (10(-8)-10(-7) M), and the adenosine agonist with higher A(1)-receptor affinity, R-N(6)-phenylisopropyladenosine (R-PIA) (10(-7), 3.2 x 10(-7), and 10(-6) M) elicited a similar degree of incremental increase of microcirculatory flow in a dose-dependent manner. The ATP-dependent potassium (K(ATP)) channel blocker glibenclamide (3.2 x 10(-6) M) significantly attenuated the vasodilation effects of these agonists. Adenosine-induced vasodilation could be significantly attenuated by the nonselective adenosine antagonist 8-(p-sulfophenyl)-theophylline (3 x 10(-5) M) or the selective A(2A) antagonist 4-(2-[7-amino-2-(2-furyl)[1,2, 4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol (ZM-241385, 10(-6) M), but not by the selective A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (5 x 10(-8) M). Adenylate cyclase inhibitor N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A, 10(-5)M) effectively suppressed the vasodilator response of adenosine and forskolin. These results suggest that adenosine-induced vasodilation in rat diaphragm microcirculation is mediated through the stimulation of A(2A) receptors, which are coupled to adenylate cyclase activation and opening of the K(ATP) channel.     

Receptor subtypes mediating adenosine-induced dilation of cerebral arterioles

The purpose of this study was to investigate the receptor subtypes that mediate the dilation of rat intracerebral arterioles elicited by adenosine. Penetrating arterioles were isolated from the rat brain, cannulated with the use of a micropipette system, and luminally pressurized to 60 mmHg. Both adenosine and the A2A receptor-selective agonist CGS-21680 induced dose-dependent vasodilation (-logEC(50): 6.5 +/- 0.2 and 8.6 +/- 0.3, respectively). However, adenosine, which is capable of activating both A2A and A2B receptors, caused a greater maximal dilation than CGS-21680. The A2A receptor-selective antagonist ZM-241385 (0.1 microM) only partially inhibited the dilation induced by adenosine but almost completely blocked CGS-21680-induced dilation. Neither 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM), an A1 receptor-selective antagonist, nor MRS-1191 (0.1 microM), an A3 receptor-selective antagonist, attenuated adenosine dose responses. Moreover, ZM-241385 had no effect on the dilation induced by ATP (10 microM) or acidic (pH 6.8) buffer. We concluded that the A2A receptor subtype mediates adenosine-induced dilation of intracerebral arterioles in the rat brain. Furthermore, our results suggest that A2B receptors may also participate in the dilation response to adenosine.     

Adenosine activation of A(2B) receptor(s) is essential for stimulated epithelial ciliary motility and clearance

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 μM) and/or procaterol (10 μM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 μM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 μM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 μM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.     

Gene transfer of Cu/Zn SOD to cerebral vessels prevents FPI-induced CBF autoregulatory dysfunction

The goal of this study was to determine whether gene transfer of human copper-zinc (Cu/Zn) superoxide dismutase (SOD) has preventive effects on cerebral blood flow (CBF) autoregulatory dysfunction after fluid percussion injury (FPI). Rats subjected to FPI (2-2.5 atm) exhibited enhanced activity of reduced NADP (NADPH) oxidase in the cerebral vasculature. In line with these findings, the rats showed not only reduced vasodilation of the pial artery in response to calcitonin gene-related peptide and levcromakalim but also impaired autoregulatory vasodilation in response to acute hypotension. The FPI-induced hemodynamic alterations were significantly prevented by pretreatment with diphenyleneiodonium (10 micromol/l), an NAD(P)H oxidase inhibitor. Intracisternal application of recombinant adenovirus (100 microl of 1 x 10(10) pfu/ml)-encoding human Cu/Zn SOD 3 days before FPI prevented the impairment of vasodilation to hypotension and vasorelaxants, resulting in the restoration of CBF autoregulation. Our findings demonstrate that FPI-induced impairment of CBF autoregulation is closely related with NAD(P)H oxidase-derived superoxide anion, and these alterations can be prevented by the recombinant adenovirus-mediated transfer of human Cu/Zn SOD gene to the cerebral vasculature.     

Effect of adenosine receptor blockade on pial arteriolar dilation during sciatic nerve stimulation

In the present study, we report the effects of adenosine receptor antagonists on pial vasodilatation during contralateral sciatic nerve stimulation (SNS). The pial circulation was observed through a closed cranial window in alpha-chloralose-anesthetized rats. In artificial cerebrospinal fluid (CSF), SNS resulted in a 30.5 +/- 13.2% increase in pial arteriolar diameter in the hindlimb somatosensory cortex. Systemic administration of the selective adenosine A2A receptor antagonist, 4-(2-[7-amino-2-[2-furyl][3,2,4]triazolol[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), significantly (P < 0.05, n = 6) attenuated the SNS-induced vasodilatation. Systemic administration of 8-(p-sulfophenyl)theophylline (8SPT), a nonselective antagonist that is blood-brain barrier (BBB) impermeable, had no effect on vasodilatation to SNS. In contrast, systemic theophylline, which readily penetrates the BBB, nearly abolished the SNS-induced vasodilatation (P < 0.01; n = 7). Topical superfusion of 8SPT significantly (P < 0.01; n = 6) attenuated vasodilatation during SNS. Topical superfusion of 8- cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, significantly potentiated SNS-induced vasodilatation (P < 0.01; n > or = 5). Hypercarbic vasodilatation and somatosensory-evoked potentials were not affected by any of the compounds tested. Our findings suggest that luminal endothelial adenosine receptors are not involved in the arteriolar response to SNS, as demonstrated by a lack of effect with systemic 8SPT. Furthermore, the adenosine A2A receptor subtype appears to be involved in the dilator response to SNS. Finally, the neuromodulatory action of adenosine, via the A1 receptor subtype, significantly influences SNS-induced vasodilatation. Thus the present study provides further evidence for a role of adenosine in the regulation of CBF during somatosensory stimulation.     

Age-related changes in adenosine-mediated relaxation of coronary and aortic smooth muscle

We tested whether adenosine mediates nitric oxide (NO)-dependent and NO-independent dilation in coronary and aortic smooth muscle and whether age selectively impairs NO-dependent adenosine relaxation. Responses to adenosine and the relatively nonselective analog 5'-N-ethylcarboxamidoadenosine (NECA) were studied in coronary vessels and aortas from immature (1-2 mo), mature (3-4 mo), and moderately aged (12-18 mo) Wistar and Sprague-Dawley rats. Adenosine and NECA induced biphasic concentration-dependent coronary vasodilation, with data supporting high-sensitivity (pEC(50) = 5.2-5.8) and low-sensitivity (pEC(50) = 2.3-2.4) adenosine sites. Although sensitivity to adenosine and NECA was unaltered by age, response magnitude declined significantly. Treatment with 50 microM N(G)-nitro-L-arginine methyl ester (L-NAME) markedly inhibited the high-sensitivity site, although response magnitude still declined with age. Aortic sensitivity to adenosine declined with age (pEC(50) = 4.7 +/- 0.2, 3.5 +/- 0.2, and 2.9 +/- 0.1 in immature, mature, and moderately aged aortas, respectively), and the adenosine receptor transduction maximum also decreased (16.1 +/- 0.8, 12.9 +/- 0.7, and 9.6 +/- 0.7 mN/mm(2) in immature, mature, and moderately aged aortas, respectively). L-NAME decreased aortic sensitivity to adenosine in immature and mature tissues but was ineffective in the moderately aged aorta. Data collectively indicate that 1) adenosine mediates NO-dependent and NO-independent coronary and aortic relaxation, 2) maturation and aging reduce NO-independent and NO-dependent adenosine responses, and 3) the age-related decline in aortic response also involves a reduction in the adenosine receptor transduction maximum.     

Protein kinase A-dependent activation of inward rectifier potassium channels by adenosine in rabbit coronary smooth muscle cells

We studied the effect of adenosine on the Ba(2+)-sensitive K(IR) channels in the smooth muscle cells isolated from the small-diameter (<100microm) coronary arteries of rabbit. Adenosine increased K(IR) currents in concentration-dependent manner (EC(50)=9.4+/-1.4microM, maximum increase of 153%). The adenosine-induced stimulation of K(IR) current was blocked by adenylyl cyclase inhibitor, SQ22536 and was mimicked by adenylyl cyclase activator, forskolin. The adenosine-induced increase of current was blocked by cyclic AMP-dependent protein kinase (PKA) inhibitors, KT 5720 and Rp-8-CPT-cAMPs. The adenosine-induced increase of K(IR) currents was blocked by an A(3)-selective antagonist MRS1334, while the antagonists of other subtypes (DPCPX for A(1), ZM241385 for A(2A), and alloxazine for A(2B)) were all ineffective. Furthermore, an A(3)-selective agonist, 2-Cl-IB-MECA induced increase of K(IR) currents. We also examined the effect of adenosine on coronary blood flow (CBF) rate by using the Langendorff-perfused heart. In the presence of glibenclamide to exclude the effects of ATP-sensitive K(+) (K(ATP)) channels, CBF was increased by adenosine (10microM), which was blocked by the addition of Ba(2+) (50microM). Above results suggest that adenosine increases K(IR) current via A(3) subtype through the activation of PKA in rabbit small-diameter coronary arterial smooth muscle cells.     

Regional differences in mechanisms of cerebral circulatory response to neuronal activation

Vibrissal stimulation raises cerebral blood flow (CBF) in the ipsilateral spinal and principal sensory trigeminal nuclei and contralateral ventroposteromedial (VPM) thalamic nucleus and barrel cortex. To investigate possible roles of adenosine and nitric oxide (NO) in these increases, local CBF was determined during unilateral vibrissal stimulation in unanesthetized rats after adenosine receptor blockade with caffeine or NO synthase inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole (7-NI). Caffeine lowered baseline CBF in all structures but reduced the percent increase during stimulation only in the two trigeminal nuclei. L-NAME and 7-NI lowered baseline CBF but reduced the percent increase during stimulation only in the higher stations of this sensory pathway, i.e., L-NAME in the VPM nucleus and 7-NI in both the VPM nucleus and barrel cortex. Combinations of caffeine with 7-NI or L-NAME did not have additive effects, and none alone or in combination completely eliminated functional activation of CBF. These results suggest that caffeine-sensitive and NO-dependent mechanisms are involved but with different regional distributions, and neither fully accounts for the functional activation of CBF.     

Impaired A2A adenosine receptor/nitric oxide/VEGF signaling pathway in fetal endothelium during late- and early-onset preeclampsia

To investigate whether fetal endothelial cell proliferation and migration are modulated by the A_2A adenosine receptor (A_2AAR), nitric oxide (NO) and the vascular endothelial growth factor (VEGF) signaling pathway, we isolated human umbilical vein endothelial cells from normal pregnancy (n?=?23), preterm delivery (n?=?4), and late-onset (LOPE, n?=?10) and early-onset preeclampsia (EOPE, n?=?8). We used the non-selective adenosine receptor agonist (NECA) and the selective agonist (CGS-21680) and/or selective antagonist (ZM-241385) for A_2AAR. Also, the nitric oxide synthase (NOS) inhibitor, l-NAME, was used in co-incubation with CGS-21680. Compared to normal pregnancy, EOPE exhibited low cell proliferation and migration associated with reduced expressions of A_2AAR and VEGF and NO synthesis (i.e., total and phosphorylated serine¹¹⁷⁷ endothelial NOS and nitrite formation). In contrast, LOPE exhibited the opposite behavior in all these markers compared to normal pregnancy or EOPE. Cell proliferation and migration were increased by CGS-21680 (or NECA) in all analyzed groups (EOPE>LOPE>normal pregnancy) compared to their respective basal conditions, an effect that was associated with high NO and VEGF synthesis and blocked by ZM-241385 with significantly different IC₅₀ for each group (EOPE>LOPE>normal pregnancy). The differences seem independent of gestational age. l-NAME blocked the CGS-21680-mediated cell proliferation and migration in normal pregnancy and LOPE (IC₅₀?=?36.2?±?2.5 and 8.6?±?2.2 nM, respectively) as well as the VEGF expression in normal pregnancy. Therefore, the A_2AAR/NO/VEGF signaling pathway exhibits a pro-angiogenic effect in normal pregnancies and LOPE, whereas impairment in this pathway seems related to the reduced angiogenic capacity of the fetal endothelium in EOPE.     

Evidence for the involvement of nitric oxide in A2B receptor-mediated vasorelaxation of mouse aorta

We have investigated the role of adenosine and its analogs on vasorelaxation of mouse aorta in intact endothelium with rank order of potency as follows: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > adenosine > CGS-21680, which is consistent with the profile of A(2B)-adenosine receptor (A(2B)AR). In endothelium-intact tissues, acetylcholine produced relaxation ranging from 65 to 80% in phenylephrine (PE, 10(-7) M)-precontracted mouse aorta, whereas no relaxation was observed in endothelium-denuded tissues. The A(2B)AR antagonist alloxazine (10(-5) M) shifted concentration-response curve for NECA (EC(50) = 0.005 x 10(-5) M) to the right with an EC(50) of 2.8 x 10(-5) M, demonstrating that this relaxation is partially dependent on functional endothelium mediated predominantly via A(2B)AR in this tissue. This conclusion was further supported by the following findings: 1) in the endothelium-intact mouse aorta, the EC(50) values for NECA and adenosine were found to be 0.05 and 1.99 x 10(-4) M, respectively; however, in denuded endothelium, these values were 0.098 and 3.55 x 10(-4) M, respectively; 2) NECA-induced relaxation was significantly blocked by N(G)-nitro-l-arginine methyl ester (l-NAME; 10(-4) M) in endothelium-intact tissues, which was reversed by pretreatment with l-arginine (10(-4) M), whereas no significant inhibition was found in endothelium-denuded tissues; 3) total nitrites and nitrates (NOx) in intact endothelium with l-NAME (10(-4) M) alone and in combination with l-arginine were 59% (P < 0.05) and 96%, respectively, in comparison with control (PE + NECA); and 4) endothelial nitric oxide synthase gene expression was found to be 67% (P < 0.05) less in endothelium-denuded as opposed to endothelium-intact mouse aorta. Thus these data demonstrate that adenosine-mediated vasorelaxation is partially dependent on A(2B)AR in mouse aorta.     

Th1 cytokines regulate adenosine receptors and their downstream signaling elements in human microvascular endothelial cells

We and others have shown that adenosine, acting at its receptors, is a potent modulator of inflammation and angiogenesis. To better understand the regulation of adenosine receptors during these processes we studied the effects of IL-1, TNF-alpha, and IFN-gamma on expression and function of adenosine receptors and select members of their coupling G proteins in human dermal microvascular endothelial cells (HMVEC). HMVEC expressed message and protein for A(2A) and A(2B), but not A(1) or A(3) receptors. IL-1 and TNF-alpha treatment increased message and protein expression of A(2A) and A(2B) receptor. IFN-gamma treatment also increased the expression of A(2B) receptors, but decreased expression of A(2A) receptors. Resting HMVEC and IFN-gamma-treated cells showed minimal cAMP response to the selective A(2A) receptor agonist 2-[2-(4-chlorophenyl)ethoxy]adenosine (MRE0094). In contrast, MRE0094 stimulated a dose-dependent increase in cAMP levels in TNF-alpha-treated cells that was almost completely blocked by the A(2A) receptor antagonist ZM-241385 (4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl]phenol). The nonselective adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine increased cAMP levels in both TNF-alpha- and IFN-gamma-treated cells, but not control cells, and its effect was only partially reversed by ZM-241385 in TNF-alpha-treated cells and not affected in IFN-gamma-treated cells. HMVEC expressed a higher level of G protein beta1 isoform than beta4 isoform. Although none of the cytokines tested affected G(beta1) expression, both IL-1 and TNF-alpha significantly up-regulated G(beta4) expression. These findings indicate that inflammatory cytokines modulate adenosine receptor expression and function on HMVECs and suggest that the interaction between proinflammatory cytokines and adenosine receptors may affect therapeutic responses to anti-inflammatory drugs that act via adenosine-dependent mechanisms.     

ATP inhibits pump activity of lymph vessels via adenosine A1 receptor-mediated involvement of NO- and ATP-sensitive K+ channels

We examined the effects of ATP on intrinsic pump activity in lymph vessels isolated from the rat. ATP caused significant dilation with a cessation of lymphatic pump activity. Removal of the endothelium or pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME) significantly reduced ATP-induced inhibitory responses of lymphatic pump activity, whereas reduction was not suppressed completely by 10(-6) M ATP. L-arginine significantly restored ATP-induced inhibitory responses in the presence of L-NAME. ATP-induced inhibitory responses in lymph vessels with endothelium were also significantly, but not completely, suppressed by pretreatment with glibenclamide. 8-Cyclopentyl-1,3-dipropylxanthine (a selective adenosine A1 receptor antagonist), but not suramine (a P2X and P2Y receptor antagonist) or 3,7-dimethyl-1-proparglyxanthine (a selective adenosine A2 receptor antagonist), significantly decreased ATP-induced inhibitory responses. alpha,beta-methylene ATP (a selective P2X and P2Y receptor agonist) had no significant effect on lymphatic pump activity. In some lymph vessels with endothelium (24 of 30 preparations), adenosine also caused dose-dependent dilation with a cessation of lymphatic pump activity. L-NAME significantly reduced the inhibitory responses induced by the lower (3 x 10(-8)-3 x 10(-7) M) concentrations of adenosine. Glibenclamide or 8-cyclopentyl-1,3-dipropylxanthine also significantly suppressed adenosine-induced inhibitory responses. These findings suggest that ATP-induced dilation and inhibition of pump activity of isolated rat lymph vessels are endothelium-dependent and -independent responses. ATP-mediated inhibitory responses may be, in part, related to production of endogenous nitric oxide, involvement of ATP-sensitive K+ channels, or activation of adenosine A1 receptors in lymphatic smooth muscle and endothelium.     

Adenosine A(2a)-receptor activation increases contractility in isolated perfused hearts

Adenosine A(2a)-receptor activation enhances shortening of isolated cardiomyocytes. In the present study the effect of A(2a)-receptor activation on the contractile performance of isolated rat hearts was investigated by recording left ventricular pressure (LVP) and the maximal rate of LVP development (+dP/dt(max)). With constant-pressure perfusion, adenosine caused concentration-dependent increases in LVP and +dP/dt(max), with detectable increases of 4.1 and 4.8% at 10(-6) M and maximal increases of 12.0 and 11.1% at 10(-4) M, respectively. The contractile responses were prevented by the A(2a)-receptor antagonists chlorostyryl-caffeine and aminofuryltriazolotriazinyl-aminoethylphenol (ZM-241385) but were not affected by the beta(1)-adrenergic antagonist atenolol. The adenosine A(1)-receptor antagonist dipropylcyclopentylxanthine and pertussis toxin potentiated the positive inotropic effects of adenosine. The A(2a)-receptor agonists ethylcarboxamidoadenosine and dimethoxyphenyl-methylphenylethyl-adenosine also enhanced contractility. With constant-flow perfusion, 10(-5) M adenosine increased LVP and +dP/dt(max) by 5.5 and 6.0%, respectively. In the presence of the coronary vasodilator hydralazine, adenosine increased LVP and +dP/dt(max) by 7.5 and 7.4%, respectively. Dipropylcyclopentylxanthine potentiated the adenosine contractile responses with constant-flow perfusion in the absence and presence of hydralazine. These increases in contractile performance were also antagonized by chlorostyryl-caffeine and ZM-241385. The results indicate that adenosine increases contractile performance via activation of A(2a) receptors in the intact heart independent of beta(1)-adrenergic receptor activation or changes in coronary flow.