Non-selective Adenosine Receptor Agonist Research Articles

Adenosine A1 Receptors Mediate Mobilization of Calcium in Human Bronchial Smooth Muscle Cells

Adenosine stimulates contraction of airway smooth muscle, but the mechanism is widely considered indirect, depending on release of contractile agonists from mast cells and nerves. The goal was to determine whether adenosine, by itself, directly regulates calcium signaling in human bronchial smooth muscle cells (HBSMC). Primary cultures of HBSMC from normal subjects were loaded with fura 2-AM, and cytosolic calcium concentrations ([Ca(2+)](i)) were determined ratiometrically by imaging single cells. The nonselective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine (CPA), both stimulated rapid, transient increases in [Ca(2+)](i). In contrast, there were no calcium responses to 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (100 nM) or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (100 nM), selective agonists at adenosine A(2A) receptors and adenosine A(3) receptors, respectively. Calcium responses to NECA and CPA were inhibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A(1) receptor antagonist, and by pertussis toxin (PTX). In other experiments, NECA stimulated calcium transients in the absence of extracellular calcium, but not when cells were preincubated in cyclopiazonic acid or thapsigargin to empty intracellular calcium stores. Calcium responses were attenuated by xestospongin C and 2-aminoethoxydiphenylborane, inhibitors of inositol trisphosphate (IP(3)) receptors, and by U73122, an inhibitor of phospholipase C. It was concluded that stimulation of adenosine A(1) receptors on HBSMC rapidly mobilizes intracellular calcium stores by a mechanism dependent on PTX-sensitive G proteins, and IP(3) signaling. These findings suggest that, in addition to its well-established indirect effects on HBSMC, adenosine also has direct effects on contractile signaling pathways.

Purinergic substances promote murine keratinocyte proliferation and enhance impaired wound healing in mice

As membrane-bound receptors for adenosine, purines, and pyrimidines, purinoceptors are expressed in nearly all cell types throughout the mammalian organism. Previous studies showed that purinoceptors are involved in the regulation of proliferation and differentiation of most target cells. The present study was performed to elucidate their role in keratinocyte proliferation and wound healing. The expression of the mRNA of several adenosine and P2Y receptors was shown in the immortalized murine keratinocyte cell line MSC-P5 and primary cultured keratinocytes of four different mouse strains. The nonselective adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine enhanced the growth of MSC-P5 cells in vitro via the A2B receptor. The proliferative stimulus of adenosine triphosphate and uridine triphosphate on this cell line was mediated by the P2Y2 receptor. The mitogenic effect of the purinergic substances was inhibited by simultaneous treatment with respective antagonists. Studies in a mouse model of dexamethasone-induced impaired wound healing showed the in vivo efficacy of the purinoceptor agonists. These studies confirm that pharmacological actions via purinoceptors offer an intriguing possibility in the treatment of impaired wound healing. Nevertheless, further investigations are needed to elucidate fully the role of purinergic mechanisms involved in wound healing.

Adenosine-Stimulated Atrial Natriuretic Peptide Release Through A 1 Receptor Subtype

Adenosine acts as an important protector of ischemic myocardium through coronary vasodilation and the depression of cardiac contractility. The protective effect of adenosine may partly relate to the cardiac hormone atrial natriuretic peptide (ANP). The aim of the present study was to investigate the effects of adenosine and the adenosine receptor subtype on atrial hemodynamics and ANP release using isolated perfused beating rat atria. Adenosine, a nonselective adenosine receptor agonist, increased the ANP release with negative inotropism in a dose-dependent manner. Adenosine-stimulated ANP release was attenuated by a selective A1 antagonist but not A(2A) antagonist or A3 antagonist. The order of potency of the various agonists for the ANP release was A1 agonists>>A3 agonist=adenosine>A(2A) agonist. The order of potency for the negative inotropy was A1 agonists>adenosine=A(2A) agonist>A3 agonist. The negative inotropism and ANP release by a specific A1 agonist (N6-cyclopentyl-adenosine) were also attenuated by A1 antagonist but not A(2A) antagonist or A3 antagonist. Treatment with A1 agonist resulted in a decrease of cAMP contents in atria and perfusates. The agonist-stimulated ANP release was significantly attenuated in the presence of forskolin, isoproterenol 8-Br-cAMP, or an adenylyl cyclase inhibitor. These results suggest that the A1 receptor subtype is responsible for the adenosine-induced ANP release and negative inotropism through adenylyl cyclase-cAMP pathway.

Adenosine Inhibits PDGF-Induced Growth of Human Glomerular Mesangial Cells Via A 2B Receptors

The objectives of the present study were to determine whether adenosine attenuates proliferation of glomerular mesangial cells (GMCs), which adenosine receptor (AR) mediates the antimitogeneic actions of adenosine, and the cellular mechanisms by which adenosine inhibits growth of GMCs. Studies were conducted in both human and rat GMCs. Platelet-derived growth factor (PDGF)-BB (25 ng/mL) increased DNA synthesis ([3H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([3H]proline incorporation), and mitogen-activated protein kinase (MAPK) activity, and these effects were attenuated by 2-chloroadenosine (nonselective AR agonist) and 5'-N-methylcarboxamidoadenosine (MECA; nonselective AR agonist), but not by N6-cyclopentyladenosine (selective A1 AR agonist), AB-N-MECA (selective A3 AR agonist), or CGS21680 (selective A(2A) AR agonist). KF17837 (selective A(2A/B) AR antagonist) and 1,3-dipropyl-8-p-sulfophenylxanthine (nonselective AR antagonist), but not 8-cyclopentyl-1,3-dipropylxanthine (selective A1 AR antagonist), blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-MECA. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor increased both basal and PDGF-induced DNA synthesis, cell proliferation, and collagen synthesis, and the growth-inhibitory effects of 2-chloroadenosine, 5'-N-MECA, and erythro-9-(2-hydroxy-3-nonyl)adenine (inhibitor of adenosine deaminase) plus iodotubercidin (inhibitor of adenosine kinase) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. We conclude that adenosine causes inhibition of GMC growth by activating A(2B) receptors coupled to inhibition of MAPK activity. A(2B) receptors may play an important role in regulating glomerular remodeling associated with GMC proliferation. Pharmacological or molecular biologic activation of A(2B) receptors may prevent glomerular remodeling associated with glomerulosclerosis, renal disease, and abnormal growth associated with hypertension and diabetes.

Characterization of the Effects of Adenosine Receptor Agonists on Cerebral Blood Flow in Uninjured and Traumatically Injured Rat Brain using Continuous Arterial Spin-Labeled Magnetic Resonance Imaging

Hypoperfusion after traumatic brain injury may exacerbate damage. Adenosine, a vasodilator, regulates cerebral blood flow (CBF). Treatment with adenosine receptor agonists has shown benefit in experimental CNS trauma; however, their effects on CBF after injury remain undefined. We used magnetic resonance imaging to assess CBF in uninjured rats both early and at 24 h after intrahippocampal administration of either the nonselective adenosine receptor agonist 2-chloroadenosine (2-CA, 12 nmol) or the A(2A)-receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarbox-amidoadenosine (CGS 21680, 6 nmol). We also assessed the effects of these agents on cerebral metabolic rate for glucose (CMRglu). We then assessed the effect of 2-CA on CBF at 3.5 to 5 h after controlled cortical impact (CCI). Injection of 2-CA into uninjured rat brain produced marked increases in CBF in ipsilateral hippocampus and cortex versus vehicle (P<0.05); CBF increases persisted even at 24 h. Measurement of hippocampal levels of 2-CA showed persistent increases to 24 h. CGS 21680 produced even more marked global increases in CBF than seen with 2-CA (2-6-fold versus vehicle, P<0.05 in 10/12 regions of interest (ROIs)). Neither agonist altered CMRglu versus vehicle. After CCI, 2-CA increased CBF in ipsilateral hippocampal and hemispheric ROIs (P<0.05 versus vehicle), but the response was attenuated at severe injury levels. We report marked increases in CBF after injection of adenosine receptor agonists into uninjured rat brain despite unaltered CMRglu. 2-Chloroadenosine produced enduring increases in CBF in uninjured brain and attenuated posttraumatic hypoperfusion. Future studies of adenosine-related therapies in CNS injury should address the role of CBF.

Involvement of p38-mitogen-activated protein kinase in adenosine receptor-mediated relaxation of coronary artery

The purpose of this study was to explore the involvement of adenosine receptor(s) in porcine coronary artery (PCA) relaxation and to define the role of MAPK signaling pathways. Isometric tensions were recorded in denuded PCA rings. 5'-(N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine receptor agonist, induced a concentration-dependent relaxation (EC(50) = 16.8 nM) of PGF(2alpha) (10 microM)-preconstricted arterial rings. NECA-induced relaxation was completely blocked by 0.1 microM SCH-58261 (A(2A) antagonist) at lower doses (1-40 nM) but not at higher doses (80-1,000 nM). MRS-1706 (1 microM, A(2B) antagonist) was able to shift the NECA concentration-response curve to the right. CGS-21680 (selective A(2A) agonist) induced responses similarly to NECA, whereas N(6)-cyclopentyladenosine (A(1) agonist) and Cl-IB-MECA (A(3) agonist) did not. Furthermore, the effect of NECA was attenuated by the addition of SB-203580 (10 microM, p38 MAPK inhibitor) but not by PD-98059 (10 microM, MEK inhibitor). Interestingly, SB-203580 had no effect on CGS-21680-induced relaxation. Western blot analysis demonstrated that PGF(2alpha) and adenosine agonists stimulated p38 MAPK at a concentration of 40 nM in PCA smooth muscle cells. MRS-1706 (1 microM) significantly reduced NECA-induced p38 MAPK phosphorylation. Addition of NECA and SB-203580 alone or in combination inhibited PGF(2alpha)-induced p38 MAPK. Western blot data were further confirmed by p38 MAPK activity measurement using activating transcription factor-2 assay. Our results suggest that the adenosine receptor subtype involved in causing relaxation of porcine coronary smooth muscle is mainly A(2A) subtype, although A(2B) also may play a role, possibly through p38 MAPK pathway.

Adenosine receptors involved in modulation of noradrenaline release in isolated rat tail artery

Adenosine receptors involved in the modulation of noradrenaline release from postganglionic sympathetic nerves in rat tail artery were characterized by studying the effects of adenosine-receptor agonists and antagonists on electrically evoked tritium overflow (100 pulses, 5 Hz) and by immunohistochemistry. The adenosine A 1 receptor-selective agonist N 6-cyclopentyladenosine (CPA; 1–100 nM) and the non-selective adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA; 1–10 μM) decreased tritium overflow. These effects were blocked by the adenosine A 1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 30 nM). The adenosine A 2A receptor-selective agonist 2- p–(2-carboxyethyl)phenethylamino-5′- N-ethylcarboxamido adenosine (CGS 21680; 1–100 nM) enhanced tritium overflow, an effect blocked by the adenosine A 2A receptor-selective antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3- e]-1,2,4-triazolo[1,5- c]pyrimidine (SCH 58261; 20 nM) but not changed by the adenosine A 2B receptor-selective antagonist N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1 H-purin-8-yl) phenoxy]acetamide (MRS 1706; 20 nM). In the presence of DPCPX (30 nM), NECA enhanced tritium overflow, an effect abolished by MRS 1706 but not influenced by SCH 58261. Immunohistochemistry revealed immunoreactivity for all adenosine-receptor subtypes. Areas of co-localization were found for neurofilament with adenosine A 1, A 2A and A 2B but not A 3 receptors. In conclusion, the present study provides functional and morphological evidence for the occurrence of multiple adenosine receptor-mediated modulation of noradrenaline release in the rat tail: inhibition mediated by adenosine A 1 receptors and facilitation mediated by both adenosine A 2A and A 2B receptors.

Coupling to protein kinases A and C of adenosine A 2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1–30 μM), but not the A 2A agonist CGS 21680 (0.001–10 μM), caused a facilitation of electrically evoked noradrenaline release (up to 43±4%), when inhibitory adenosine A 1 receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A 2B receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of α 1-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A 2B receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase–protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A 2B receptor-mediated facilitation of noradrenaline release.

Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1–30 μM), but not the A 2A agonist CGS 21680 (0.001–10 μM), caused a facilitation of electrically evoked noradrenaline release (up to 43±4%), when inhibitory adenosine A 1 receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A 2B receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of α 1 -adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A 2B receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase–protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A 2B receptor-mediated facilitation of noradrenaline release.

Effects of synthetic adenosine receptor agonists on growth characteristics of G:5:113 fibrosarcoma cells In Vitro

AbstractThe effects of several synthetic adenosine receptor agonists on the growth characteristics of murine G:5:113 fibrosarcoma cells in vitro were evaluated. Cell number and viability, as well as proportions of cells in individual cell cycle phases, were determined after 48 h cultivation of the cells in the presence of concentrations of adenosine receptor agonists ranging between 0.01 and 500 µM. Effects of the non‐selective adenosine receptor agonist NECA, as well as of agonists selective for A1 (CPA), A2A (CGS 21680), and A3 receptors (IB‐MECA) were evaluated. A slight increase in cell number was observed at a narrow range of concentrations between 30–50 µM of NECA, which was reproduced by CPA, a selective A1 receptor agonist, in concentrations ranging from 20–50 µM. The most important finding was that of significant inhibitory effects of adenosine receptor agonists on cell number in concentrations above 50 µM. This effect was most pronounced after treatment of the G:5:113 fibrosarcoma cells with the selective A3 receptor agonist, IB‐MECA, as revealed also by calculated values of generation time and EC50. The results suggest that adenosine A3 receptor might represent a target for pharmacological anticancer approaches aimed at suppressing the growth of fibrosarcoma cell population. Drug Dev. Res. 60:303–311, 2003. © 2003 Wiley‐Liss, Inc.

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.

Functional expression of adenosine A 2A and A 3 receptors in the mouse dendritic cell line XS-106

There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A 1, A 2A and A 3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A 3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9 H-purin-9-yl]-1-deoxy- N-methyl-β- d-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [ 3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A 2A receptor agonist 4-[2-[[-6-amino-9-( N-ethyl-β- d-ribofuranuronamidosyl)-9 H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [ 3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A 1 receptor agonist CPA ( N 6-cyclopentyladenosine) did not inhibit forskolin-mediated [ 3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A 2A and A 3 receptors. The non-selective adenosine receptor agonist 5′- N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-α (TNF-α) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 μM) or CGS 21680 (1 μM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-α release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-α release comparable to that observed with NECA alone. Treatment of cells with the adenosine A 2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3- a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385; 100 nM) and 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo[4,3- e]-1,2,4-triazolo[1,5 c]pyrimidine (SCH 58261; 100 nM) and the adenosine A 3 receptor selective antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5- c]quinazolin-5-benzeneacetamide (MRS 1220; 100 nM) partially blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-α release. Combined addition of MRS 1220 and SCH 58261 completely blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-α release. In conclusion, we have shown that the mouse dendritic cell line XS-106 expresses functional adenosine A 2A and A 3 receptors, which are capable of modulating TNF-α release.

Regional differences in the adenosine A 2 receptor-mediated modulation of contractions in rat vas deferens

Adenosine receptors involved in modulation of contractions were characterized in the bisected rat vas deferens by combining pharmacological and immunohistochemical approaches. In both portions, noradrenaline-elicited contractions were enhanced by the adenosine A 1 receptor agonist N 6-cyclopentyladenosine (CPA), and inhibited by the non-selective adenosine receptor agonist 5′- N-ethylcarboxamidoadenosine (NECA) in the presence of the adenosine A 1 receptor antagonist 1,3-dipropyl-8-cyclopentyl-l,3-dipropylxanthine (DPCPX). The adenosine A 2A receptor agonist 2- p-(2-carboxyethyl)phenethyl-amino-5′- N-ethylcarboxamidoadenosine (CGS 21680) also inhibited noradrenaline-elicited contractions but only in the prostatic portion. Contractions elicited by the stable ATP analogue α,β-methyleneATP (α,β-MeATP) were inhibited only by NECA in the presence of DPCPX and only in the prostatic portion. This study provides functional evidence for the presence, in both portions of the rat vas deferens, of an adenosine A 1 receptor-mediated enhancement and of an adenosine A 2 receptor-mediated inhibition of contractions. The latter effect is mediated by both A 2A and A 2B subtypes in the prostatic portion but only by the A 2B subtype in the epididymal portion. This regional variation is supported by the immunohistochemical results that revealed an adenosine A 2A receptor immunoreactivity not co-localized with nerve fibres more abundant in the prostatic than in the epididymal portion.

Involvement of K+ channels in adenosine A2A and A2B receptor-mediated hyperpolarization of porcine coronary artery endothelial cells.

This study investigated the effects of the following adenosine agonists: 5;-ethylcarboxamidoadenosine (NECA), N6-cyclopentyadenosine (CPA) 2-[p-(2-carboxyethyl)]phenylamino-5;N-ethylcarboxamidoadenosine (CGS-21680), and 2-chloroadenosine (CAD) and its 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), a selective A2A adenosine receptor antagonist, and the involvement of the K+ATP and KCa channels on the resting membrane potential (RMP) of confluent monolayers of cultured porcine coronary artery endothelial cells (PCAECs). Adenosine agonists and K+ATP channel openers (pinacidil, cromakalim) hyperpolarized cultured PCAECs. The average RMP was -32.31 +/- 1.2 mV. Adenosine agonists at 10-5 M caused a significant increase in RMP to -65.0 +/- 1.5 mV for CAD (a nonselective adenosine receptor agonist) to -75.9 +/- 1.6 mV for CGS-21680 (a selective A2A receptor agonist) and to -87.0 +/- 3.5 mV for NECA (a nonselective A1/A2A/A2B receptor agonist). Pinacidil and cromakalim at 10 microM increased the membrane potential to -76.2 +/- 1.2 mV and -75.22 +/- 0.12 mV, respectively. The hyperpolarization induced by adenosine receptor agonists and KATP openers was inhibited by an application of the K+ATP channel blocker glibenclamide (10 microM), indicating the involvement of the K+ATP channel in the adenosine-mediated hyperpolarization of PCAECs. Moreover, 1-EB10, a selective opener of the maxi-KCa channel, hyperpolarized PCAECs, and the effect of 1-EB10 was completely blocked by a selective, irreversible blocker of the high conductance KCa (maxi-K) channels (penitrem A), but it only partially blocked the effect of NECA. ZM-241385 has no effect on hyperpolarization elicited by K+ATP and KCa channel openers. However, ZM-241385 significantly blocked the hyperpolarization effect of CAD and CGS-21680. ZM-241385 partially blocked the hyperpolarizing effect of NECA, and a combination of ZM-241385 and penitrem A further blocked the hyperpolarizing effect of NECA. These results further support the involvement of K+ channels in adenosine A2A and A2B receptor-mediated hyperpolarization of PCAECs.

Inhibition of NF-kappaB-mediated gene transcription by the human A2B adenosine receptor in Chinese hamster ovary cells.

NF-kappaB is a transcription factor that plays a vital role in regulating inducible gene expression in immune and inflammatory responses. In view of the well documented effects of adenosine on immune and inflammatory responses, we have explored whether adenosine A1, A2B and A3 receptors regulate NF-kappaB activity in transfected Chinese hamster ovary (CHO) cells using a luciferase reporter gene construct. No increases in NF-kappaB activity were observed in CHO-A1, -A2B and -A3 cells stimulated with the non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine. Elevating intracellular cyclic AMP (cAMP) levels using forskolin (direct activator of adenylyl cyclase) and rolipram (type IV, cAMP-specific phosphodiesterase inhibitor), inhibited NF-kappaB activity in CHO cells. Adenosine A2B receptor stimulation also inhibited NF-kappaB activity, whereas adenosine A1 and A3 receptor activation had no effect. These data reflect the known coupling of adenosine A2B receptors to increases in cAMP. In conclusion, adenosine A1, A2B and A3 receptors do not directly activate NF-kappaB in CHO cells. However, adenosine A2B receptor activation significantly inhibited NF-kappaB activity. Inhibition of NF-kappaB activity by the adenosine A2B receptor may contribute to the anti-inflammatory effects of adenosine.

The adenosine receptor agonist, APNEA, increases calcium influx into rat cortical synaptosomes through N-type channels associated with A2a receptors.

N6-2-(4-aminophenyl)ethyladenosine (APNEA) is a nonselective adenosine receptor agonist known to have a high affinity for the adenosine A1 and A3 receptors. It was found to be able to dose-dependently increase the sustained (4 min) Ca2+ influx into rat cortical synaptosomes while 2-chloro-N6-(3-iodobenzyl)-adenosine-5-N-methyluronamide (Cl-IB-MECA), a selective A3 agonist has no effect. However, this effect of APNEA was not affected by the presence of 8-cyclopentyl-1,3-dimethylxanthine (CPT), a selective A1 antagonist; but instead completely abolished by 8-(3-chlorostyryl)caffeine (CSC), a selective A2a antagonist, or omega-conotoxin GVIA. These results show that in the rat cortex, presynaptic A2a receptors can mediate neurotransmitter release by increasing Ca2+ influx through the N-type calcium channels. A1 and A3 receptors appear not to be involved.

Adenosine inhibits a non-inactivating K+ current in bovine adrenal cortical cells by activation of multiple P1 receptors.

1. Bovine adrenal zona fasciculata (AZF) cells express a non-inactivating K+ current (IAC) that sets the resting potential while it is activated by intracellular ATP. In whole-cell patch clamp recordings from bovine AZF cells, we found that adenosine selectively inhibited IAC by a maximum of 78.4 +/- 4.6 % (n = 8) with an IC50 of 71 nM. The non-selective adenosine receptor agonist NECA effectively inhibited IAC by 79.3 +/- 2.9 % (n = 24) at a concentration of 100 nM. 2. Inhibition of IAC was mediated through multiple P1 adenosine receptor subtypes. The A1-selective agonist CCPA (10 nM), the A2A-selective agonist CGS 21680 (100 nM) and the A3-selective agonist IB-MECA (10 nM) inhibited IAC by 64.8 +/- 8.4, 78.4 +/- 4.6 and 69.3 +/- 6.9 %, respectively. 3. Specific adenosine receptor subtype antagonists including DPCPX (A1), ZM 241385 (A2A) and MRS 1191 (A3) effectively blocked inhibition of IAC by adenosine receptor-selective agonists. 4. A mixture of the three adenosine receptor antagonists completely suppressed inhibition of IAC by adenosine, but failed to alter inhibition by external ATP which acts through a separate P2 nucleotide receptor. 5. Inhibition of IAC by adenosine or NECA was eliminated by substituting GDP-beta-S for GTP in the pipette, or by replacing ATP with AMP-PNP or UTP. 6. In addition to inhibiting IAC, adenosine (10 microM) depolarized AZF cells by 46.2 +/- 5.8 mV (n = 6). 7. These results show that bovine AZF cells express at least three adenosine receptor subtypes (A1, A2A, A3), each of which is coupled to the inhibition of IAC K+ channels through a G-protein-dependent mechanism requiring ATP hydrolysis. Adenosine-mediated inhibition of IAC is associated with membrane depolarization. Adenosine and other purines may co-ordinate the stress-induced secretion of corticosteroids and catecholamines from the adrenal gland.

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

Role of A2A receptor in the modulation of myocardial reperfusion damage.

Adenosine protects myocardium from ischemia and reperfusion damage; however, the mechanism of action is still under discussion. We investigated whether (a) adenosine protects isolated crystalloid-perfused rabbit heart from ischemia/ reperfusion injury; (b) this action is receptor mediated and what receptor subtypes are involved, and (c) this action is dependent on an enhanced nitric oxide production. Our results showed a cardioprotective effect of adenosine (10(-4) M), of nonselective adenosine-receptor agonist 5'-N-ethyl-carboxamidoadenosine (NECA; 5 x 10(-6) M), and of A2A agonists CGS 21680 (10(-8) and 10(-6) M), 2-hexynylNECA (10(-7) M). On the contrary, A1 agonist CCPA (10(-8) and 10(-6) M) does not provide any protection. The effect has been achieved in terms of significant reduction in contracture development during reperfusion [diastolic pressure was 46.8 +/- 7.1 mm Hg (p < 0.01); 46.1 +/- 7.8 mm Hg (p < 0.01); 46.9 +/- 5.5 mm Hg (p < 0.01); and 59.3 +/- 6.7 mm Hg (p < 0.05) with 10(-4) M adenosine, 5 x 10(-6) M NECA, 10(-6) M CGS 21680, and 10(-7) M 2-hexynylNECA, respectively, versus 77.6 +/- 5.0 mm Hg in control]; reduced creatine phosphokinase release (13.5 +/- 1.6, 22.2 +/- 7.9, 14.2 +/- 3.3, and 14.1 +/- 4.5 U/gww in treated hearts vs. 34.6 +/- 7.2 U/gww in controls; p < 0.05); improved energy metabolism [adenosine triphosphate (ATP) content is 9.9 +/- 0.5, 10.4 +/- 0.6, 9.8 +/- 0.5, and 10.5 +/- 0.5 micromol/gdw in treated hearts vs. 7.6 +/- 0.2 micromol/gdw; p < 0.05]. Moreover, our data indirectly show a functional presence of A2A receptors on cardiomyocytes as the protection is A2A mediated and exerted only during reperfusion, although in the absence of blood and coronary flow changes. These activities appear independent of nitric oxide pathways, as adenosine and 2-hexynylNECA effects are not affected by the presence of a nitric oxide-synthase inhibitor (10(-4) M L-NNA).

Examination of adenosine receptor-mediated relaxation of the pig coronary artery.

1. The adenosine receptors mediating relaxation of porcine isolated left anterior descending coronary arteries (LAD) and the effects of the level and type of preconstriction on the responses to adenosine analogues were examined in the present study. 2. Relaxation responses to the non-selective adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA) were endothelium independent. N-Ethylcarboxamidoadenosine, GR 79236 (A1 receptor selective) and 8-cyclopentyl-1,3-dipropylxanthine (CGS 21680) (A2A receptor selective) produced full relaxation in LAD precontracted to 50% of the response to potassium depolarization with the thromboxane receptor agonist U46619. The order of potency was CGS 21680 = NECA > GR 79236, consistent with that defining the A2A receptor subtype. 3. 3,7-Dimethyl-1-propargylxanthine (DMPX; A2 receptor selective) competitively antagonized NECA and CGS 21680 with pKB values of 4.95 +/- 0.09 and 5.06 +/- 0.22, respectively. The A1 receptor selective antagonist 1,3-[3H]-dipropyl-8-cyclopentylxanthine (DPCPX) had no effect on NECA relaxation, even in the presence of DMPX. 4. The sensitivity to relaxation by NECA was dependent on the precontracting agent. Arteries precontracted with endothelin (ET)-1 were most sensitive to NECA, U46619-precontracted arteries were intermediate and KCl-precontracted arteries were least sensitive. 5. The potency of NECA was reduced when the preconstriction level was increased from 50 to 90% of maximum in U46619-precontracted arteries (pEC50 7.94 +/- 0.12 and 7.35 +/- 0.04, respectively) and, in KCl-precontracted arteries, both the potency and maximum effect of NECA were reduced when the preconstriction level increased from 50 to 80% of maximum (pEC50 7.52 +/- 0.13 and 6.91 +/- 0.26, respectively; maximum responses 82.5 +/- 10.2 and 23.9 +/- 3.6%, respectively, of the preconstricted tone). Relaxation responses to NECA were independent of the level of precontraction in ET-1-precontracted arteries. 6. In porcine LAD, relaxation responses to adenosine analogues were endothelium independent and were mediated via A2A adenosine receptors. Responses to NECA were dependent on both the level and type of preconstriction.