Hog barn dust extract increases macromolecular efflux from the hamster cheek pouch.

The purpose of this study was to determine whether short-term exposure to an aqueous extract of hog barn dust increases macromolecular efflux from the intact hamster cheek pouch and, if so, to begin to determine the mechanism(s) underlying this response. By using intravital microscopy, we found that suffusion of hog barn dust extract onto the intact hamster cheek pouch for 60 min elicited a significant, concentration-dependent leaky site formation and increase in clearance of FITC-labeled dextran (molecular mass, 70 kDa). This response was significantly attenuated by suffusion of catalase (60 U/ml), but not by heat-inactivated catalase, and by pretreatment with dexamethasone (10 mg/kg iv) (P < 0.05). Catalase had no significant effects on adenosine-induced increase in macromolecular efflux from the cheek pouch. Suffusion of hog barn dust extract had no significant effects on arteriolar diameter in the cheek pouch. Taken together, these data indicate that hog barn dust extract increases macromolecular efflux from the in situ hamster cheek pouch, in part, through local elaboration of reactive oxygen species that are inactivated by catalase. This response is specific and attenuated by corticosteroids. We suggest that plasma exudation plays an important role in the genesis of upper airway dysfunction evoked by short-term exposure to hog barn dust.     

Dexamethasone attenuates grain sorghum dust extract-induced increase in macromolecular efflux in vivo.

The purpose of this study was to determine whether dexamethasone attenuates grain sorghum dust extract-induced increase in macromolecular efflux from the in situ hamster cheek pouch and, if so, whether this response is specific. By using intravital microscopy, we found that an aqueous extract of grain sorghum dust elicited significant, concentration-dependent leaky site formation and increase in clearance of FITC-labeled dextran (FITC-dextran; mol mass, 70 kDa) from the in situ hamster cheek pouch (P < 0.05). This response was significantly attenuated by dexamethasone (10 mg/kg iv). Dexamethasone also attenuated substance P-induced leaky site formation and increase in clearance of FITC-dextran from the cheek pouch but had no significant effects on adenosine-induced responses. Dexamethasone had no significant effects on arteriolar diameter in the cheek pouch. On balance, these data indicate that dexamethasone attenuates grain sorghum dust extract- and substance P-induced increases in macromolecular efflux from the in situ hamster cheek pouch in a specific fashion.     

Dexamethasone attenuates acute macromolecular efflux increase evoked by smokeless tobacco extract.

The purpose of this study was to determine whether dexamethasone attenuates the acute increase in macromolecular efflux from the oral mucosa elicited by an aqueous extract of smokeless tobacco (STE) in vivo, and, if so, whether this response is specific. Using intravital microscopy, we found that 20-min suffusion of STE elicited significant, concentration-related leaky site formation and an increase in clearance of fluorescein isothiocyanate-labeled dextran (FITC-dextran; mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05). This response was significantly attenuated by dexamethasone (10 mg/kg iv). Dexamethasone also attenuated the bradykinin-induced leaky site formation and the increase in clearance of FITC-dextran from the cheek pouch. However, it had no significant effects on adenosine-induced responses. Dexamethasone had no significant effects on baseline arteriolar diameter and on bradykinin-induced vasodilation in the cheek pouch. Collectively, these data indicate that dexamethasone attenuates, in a specific fashion, the acute increase in macromolecular efflux from the in situ oral mucosa evoked by short-term suffusion of STE. We suggest that corticosteroids mitigate acute oral mucosa inflammation elicited by smokeless tobacco.     

Bradykinin- and substance P-induced edema formation in the hamster cheek pouch is tyrosine kinase dependent.

The purpose of this study was to determine whether protein tyrosine kinase, a ubiquitous family of intracellular signaling enzymes that regulates endothelial cell function, modulates bradykinin- and substance P-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation. Using intravital microscopy, I found that suffusion of bradykinin or substance P (each, 0.5 and 1.0 microM) onto the cheek pouch elicited significant, concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-dextran (FITC-dextran; molecular mass, 70 kDa; P < 0.05). These responses were significantly attenuated by suffusion of genistein (1.0 microM) or tyrphostin 25 (10 microM), two structurally unrelated, nonspecific protein tyrosine kinase inhibitors (P < 0.05). Conceivably, the kinase(s) involved in this process could be agonist specific because genistein was more effective than tyrphostin 25 in attenuating bradykinin-induced responses while the opposite was observed with substance P. Both inhibitors had no significant effects on adenosine (0.5 M)-induced responses (P > 0.5). Collectively, these data suggest that the protein tyrosine kinase metabolic pathway modulates, in part, the edemagenic effects of bradykinin and substance P in the intact hamster cheek pouch microcirculation in a specific fashion.     

Targeted deletion of adenosine A(3) receptors augments adenosine-induced coronary flow in isolated mouse heart

To determine whether adenosine A(3) receptors participate in adenosine-induced changes in coronary flow, isolated hearts from wild-type (WT) and A(3) receptor knockout (A(3)KO) mice were perfused under constant pressure and effects of nonselective and selective agonists were examined. Adenosine and the selective A(2A) agonist 2-[p-(2-carboxyethyl)]phenylethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) produced augmented maximal coronary vasodilation in A(3)KO hearts compared with WT hearts. Selective activation of A(3) receptors with 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) at nanomolar concentrations did not effect coronary flow, but at higher concentrations it produced coronary vasodilation both in WT and A(3)KO hearts. Cl-IB-MECA-induced increases in coronary flow were susceptible to both pharmacological blockade and genetic deletion of A(2A) receptors. Because deletion or blockade of adenosine A(3) receptors augmented coronary flow induced by nonselective adenosine and the selective A(2A) receptor agonist CGS-21680, we speculate that this is due to removal of an inhibitory influence associated with the A(3) receptor subtype. These data indicate that the presence of adenosine A(3) receptors may either inhibit or negatively modulate coronary flow mediated by other adenosine receptor subtypes.     

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.     

Tyrosine kinase inhibitors modulate agonist-induced vasodilation in the hamster cheek pouch.

The purpose of this study was to determine whether inhibitors of tyrosine kinase attenuate vasodilation elicited by endogenously elaborated and exogenously applied nitric oxide in the in situ peripheral microcirculation. Using intravital microscopy, we found that pretreatment with genistein (1.0 microM) and tyrphostin 25 (10.0 microM), two structurally unrelated tyrosine kinase inhibitors, significantly attenuated acetylcholine-, bradykinin- and nitroglycerin-induced dilation of second-order arterioles (51 +/- 1 microm) in the in situ hamster cheek pouch (P < 0.05). Both inhibitors nearly abrogated acetylcholine-induced responses but only partially blocked bradykinin- and nitroglycerin-induced vasodilation. Genistein and tyrphostin 25 alone had no significant effects on resting arteriolar diameter and on adenosine-induced vasodilation in the cheek pouch. On balance, these data indicate that tyrosine kinase inhibitors attenuate endogenously elaborated and exogenously applied nitric oxide-induced vasodilation in the in situ hamster cheek pouch. However, the extent of tyrosine kinase inhibitor-sensitive pathway involvement in this response appears to be agonist dependent.     

Purified ACE attenuates smokeless tobacco-induced increase in macromolecular efflux from the oral mucosa

Gao, Xiao-pei, Hideyuki Suzuki, Christopher O. Olopade,Sergei Pakhlevaniants, and Israel Rubinstein. Purified ACE attenuates smokeless tobacco-induced increase in macromolecular effluxfrom the oral mucosa. J. Appl.Physiol. 83(1): 74-81, 1997.The purpose of thisstudy was to determine whether purified angiotensin I-converting enzyme(ACE) attenuates smokeless tobacco extract (STE)-induced increase inmacromolecular efflux from the in situ oral mucosa. Byusing intravital microscopy, we found that suffusion of an aqueousextract of smokeless tobacco elicited significant concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-labeled dextran (mol mass, 70 kDa) fromthe hamster cheek pouch (P < 0.05). Suffusion of purified rabbit lung ACEsignificantly attenuated these responses in a concentration-dependentfashion (P < 0.05). These effectswere specific because purified ACE also significantly attenuated the increase in macromolecular efflux elicited by bradykinin, which isproduced in the cheek pouch during suffusion of STE, but did notattenuate the increase elicted by adenosine. Moreover,suffusion of heat-inactivated purified ACE and purified superoxidedismutase had no significant effects on STE- andbradykinin-induced responses. Collectively, these data suggestthat exogenous ACE attenuates STE-induced increase in macromolecularefflux from the in situ oral mucosa, in part, by promoting localbradykinin catabolism.

     

Beneficial effects of adenosine A(2a) agonist CGS-21680 in infarcted and stunned porcine myocardium

Although there are conflicting results on whether adenosine infusion during reperfusion alters infarct size, there are several reports that indicate adenosine A(2a) agonists reduce infarct size. There are also reports that the A(2a) agonist CGS-21680 increases cAMP and contractility in ventricular myocytes. The purpose of this study was to determine whether low-dose intracoronary infusions of CGS-21680 during reperfusion exert any beneficial effects in irreversibly and reversibly injured myocardium. Open-chest pigs were submitted to 60 min of coronary artery occlusion and 3 h of reperfusion. Treated pigs were administered intracoronary CGS-21680 (0.2 microg x kg(-1) x min(-1)) for the first 60 min of reperfusion. Pigs submitted to regional stunning (15 min ischemia) were treated with intracoronary CGS-21680 (0.15 microg x kg(-1) x min(-1)) after 2 h of reperfusion. In the infarct protocol, CGS-21680 reduced infarct size from 62 +/- 2% of the region at risk to 36 +/- 2%. In stunned myocardium, CGS increased load-independent regional preload recruitable stroke work and area by > or =70%, but the same infusion in normal myocardium was associated with no inotropic effect. Both beneficial effects were associated with little systemic hemodynamic effects. These findings suggest that reperfusion infusions of low doses of the A(2a) agonist CGS-21680 exert beneficial effects in reversibly and irreversibly injured myocardium.     

Effects of Adenosine Receptor Subtypes on Hippocampal Extracellular Serotonin Level and Serotonin Reuptake Activity

Abstract: To clarify the effects of adenosine receptor subtypes (A₁, A₂, and A₃) on hippocampal serotoninergic function, hippocampal extracellular serotonin (5-HT) levels were determined by in vivo microdialysis in freely moving rats under various conditions. Both adenosine and an adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyladenosine, decreased extracellular 5-HT levels, whereas an adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), and caffeine increased these levels. A selective A_2A receptor agonist (CGS-21680), an adenosine A₂ receptor agonist (PD-125944), an adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), and an adenosine A₃ receptor agonist, N⁶-2-(4-aminophenyl)ethyladenosine (APNEA), did not affect extracellular 5-HT levels. When the adenosine A₁ receptor was blocked by CPT, the hippocampal extracellular 5-HT level was increased by adenosine, CGS-21680, and PD-125944, and decreased by caffeine, DMPX, and APNEA. When both adenosine A₁ and A₂ receptors were blocked by CPT and DMPX, the extracellular 5-HT level was decreased by adenosine, caffeine, and APNEA. The hippocampal extracellular 5-HT level was not affected by administration of APNEA alone, but was decreased by this agent when the adenosine A₁ receptor was blocked, irrespective of whether the adenosine A₂ receptor was functional. These inhibitory effects of adenosine, caffeine, and APNEA on extracellular 5-HT levels, during both adenosine A₁ and A₂ receptor blockade, were inhibited by selective 5-HT reuptake inhibitors. These results indicate that the stimulatory effects of the adenosine A₂ receptor and the inhibitory effects of the A₃ receptor on hippocampal extracellular 5-HT levels are masked by the inhibitory effects of the adenosine A₁ receptor.     

GABA Alters GABAA Receptor mRNAs and Increases Ligand Binding

Abstract: Adenosine A_2a receptors have been localized to GABAergic striatopallidal neurons, but their functional role is unknown. To address this question, the modulation of endogenous GABA release by adenosine A_2a receptors was examined in slices of rat globus pallidus. The selective adenosine A_2a receptor agonist CGS-21680 (3.0–10 n M ) significantly increased electrically stimulated release (overflow) of GABA, with 10 n M CGS-21680 resulting in a 44% increase compared with the control. Both the nonselective adenosine receptor antagonist 8-phenyltheophylline (10 μ M ) and the selective A_2a receptor antagonist KF-17837 (100 n M ) abolished the CGS-21680-induced increase in GABA overflow. Higher concentrations of CGS-21680 (0.10–1.0 μ M ) decreased GABA overflow by ˜25%.8-Phenyltheophylline (10 μ M ) antagonized these effects, whereas KF-17837 (100 n M ) did not, suggesting actions of CGS-21680 on other adenosine receptors at these concentrations. These results demonstrate that activation of adenosine A_2a receptors augments electrically stimulated release of GABA from globus pallidus slices and suggest a mechanism by which adenosine may modulate GABAergic output from the striatopallidal efferent system.     

Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells

Adenosine, acting at its receptors, particularly A(2A) receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A(2A) receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective A(2A) receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A(2A) receptor 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). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-alpha (100 U/ml) but markedly attenuated in cells pretreated with IFN-gamma (100 U/ml). Similarly, IL-1 and TNF-alpha treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-gamma treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-alpha-treated cells but not in control or IFN-gamma-treated cells. Both IL-1 and TNF-alpha increased A(2A) receptor mRNA and protein. In parallel with its effect on A(2A) receptor function, IFN-gamma down-regulated A(2A) receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells.     

Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes

Adenosine (ADO) exerts potent anti-inflammatory and immunosuppressive effects. In this paper we address the possibility that these effects are partly mediated by inhibition of the secretion of IL-12, a proinflammatory cytokine and a major inducer of Th1 responses. We demonstrate that 5'-N-ethylcarboxamidoadenosine (NECA), a nonspecific ADO analogue, and 2-p-(2-carbonyl-ethyl)phenylethylamino-5'-N-ethylcarboxamidoadenos ine (CGS-21680), a specific A2a receptor agonist, dose-dependently inhibited, in whole blood ex vivo and monocyte cultures, the production of human IL-12 induced by LPS and Stapholococcus aureus Cowan strain 1. However, the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine and the A3 receptor agonists N6-Benzyl-NECA and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-d -ribofuranuronamide expressed only weak inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of IL-10. The differential effect of these drugs on monocyte IL-12 and IL-10 production implies that these effects are mediated by A2a receptor signaling rather than by intracellular toxicity of ADO analogue's metabolites. Moreover, CGS-21680 inhibited IL-12 production independently of endogenous IL-10 induction, because anti-IL-10 Abs failed to prevent its effect. The selective A2a antagonist 8-(3-Chlorostyryl) caffeine prevented the inhibitory effect of CGS-21680 on IL-12 production. The phosphodiesterase inhibitor Ro 20-1724 dose-dependently potentiated the inhibitory effect of CGS-21680 and, furthermore, Rp-cAMPS, a protein kinase A inhibitor, reversed the inhibitory effect of CGS-21680, implicating a cAMP/protein kinase A pathway in its action. Thus, ligand activation of A2a receptors simultaneously inhibits IL-12 and stimulates IL-10 production by human monocytes. Through this mechanism, ADO released in excess during inflammatory and ischemic conditions, or tissue injury, may contribute to selective suppression of Th1 responses and cellular immunity.     

Differential role of ionotropic glutamatergic mechanisms in responses to NTS P(2x) and A(2a) receptor stimulation

Activation of ATP P(2x) receptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of alpha,beta-methylene ATP (alpha,beta-MeATP) elicits fast initial depressor and sympathoinhibitory responses that are followed by slow, long-lasting inhibitory effects. Activation of NTS adenosine A(2a) receptors via microinjection of CGS-21680 elicits slow, long-lasting decreases in arterial pressure and renal sympathetic nerve activity (RSNA) and an increase in preganglionic adrenal sympathetic nerve activity (pre-ASNA). Both P(2x) and A(2a) receptors may operate via modulation of glutamate release from central neurons. We investigated whether intact glutamatergic transmission is necessary to mediate the responses to NTS P(2x) and A(2a) receptor stimulation. The hemodynamic and neural (RSNA and pre-ASNA) responses to microinjections of alpha,beta-MeATP (25 pmol/50 nl) and CGS-21680 (20 pmol/50 nl) were compared before and after pretreatment with kynurenate sodium (KYN; 4.4 nmol/100 nl) in chloralose-urethan-anesthetized male Sprague-Dawley rats. KYN virtually abolished the fast responses to alpha,beta-MeATP and tended to enhance the slow component of the neural responses. The depressor responses to CGS-21680 were mostly preserved after pretreatment with KYN, although the increase in pre-ASNA was reduced by one-half following the glutamatergic blockade. We conclude that the fast responses to stimulation of NTS P(2x) receptors are mediated via glutamatergic ionotropic mechanisms, whereas the slow responses to stimulation of NTS P(2x) and A(2a) receptors are mediated mostly via other neuromodulatory mechanisms.     

NTS A(2a) purinoceptor activation elicits hindlimb vasodilation primarily via a beta-adrenergic mechanism

Previously, we have shown that activation of adenosine A(2a) receptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of the selective A(2a) receptor agonist CGS-21680 elicits potent, dose-dependent decreases in mean arterial pressure and preferential, marked hindlimb vasodilation. Although A(2a) receptor activation does not change lumbar sympathetic nerve activity, it does markedly enhance the preganglionic adrenal sympathetic nerve activity, which will increase epinephrine release and could subsequently elicit hindlimb vasodilation via activation of beta(2)-adrenergic receptors. Therefore we investigated whether this hindlimb vasodilation was due to neural or humoral mechanisms. In chloralose-urethan-anesthetized male Sprague-Dawley rats, we monitored cardiovascular responses to stimulation of NTS adenosine A(2a) receptors (CGS-21680, 20 pmol/50 nl) in the intact control animals; after pretreatment with propranolol (2 mg/kg iv), a beta-adrenergic antagonist; after bilateral lumbar sympathectomy; after bilateral adrenalectomy; and after combined bilateral lumbar sympathectomy and adrenalectomy. After beta-adrenergic blockade, stimulation of NTS adenosine A(2a) receptors produced a pressor response and a hindlimb vasoconstriction. Lumbar sympathectomy reduced the vasodilation seen in the intact animals by approximately 40%, and adrenalectomy reduced it by approximately 80%. The combined sympathectomy and adrenalectomy virtually abolished the hindlimb vasodilation evoked by NTS A(2a) receptor activation. We conclude that the preferential, marked hindlimb vasodilation produced by stimulation of NTS adenosine A(2a) receptors is mediated by both the efferent sympathetic nerves directed to the hindlimb and the adrenal glands via primarily a beta-adrenergic mechanism.     

Stimulation of A(₂a) adenosine receptor abolishes the inhibitory effect of arachidonic acid on the basolateral 50-pS K channel in the thick ascending limb

The basolateral 50-pS K channels are stimulated by a cAMP-dependent pathway and inhibited by cytochrome P-450-omega-hydroxylase-dependent metabolism of arachidonic acid (AA) in the rat thick ascending limb (TAL). We now used the patch-clamp technique to examine whether stimulation of adenosine A(?a) receptor modulates the inhibitory effect of AA on the basolateral 50-pS K channels in the medullary TAL. Stimulation of adenosine A(?a) receptor with CGS-21680 or inhibition of phospholipase A? (PLA?) with AACOCF3 increased the 50-pS K channel activity in the TAL. Western blot demonstrated that application of CGS-21680 decreased the phosphorylation of PLA(2) at serine residue 505, an indication of inhibiting PLA? activity. In the presence of CGS-21680, inhibition of PLA? had no further effect on the basolateral 50-pS K channels. The possibility that CGS-21680-induced stimulation of the basolateral 50-pS K channels was partially achieved by inhibition of PLA? in the TAL was also supported by the observation that CGS-21680 had no additional effect in the presence of AACOCF3. Moreover, stimulation of adenosine A(?a) receptor with CGS-21680 also abolished the inhibitory effect of AA and 20-hydroxyeicosatetraenoic acid (20-HETE) on the 50-pS K channels. The effect of CGS-21680 on AA and 20-HETE-mediated inhibition of the 50-pS K channels was mediated by cAMP because application of membrane-permeable cAMP analog, dibutyryl-cAMP, not only increased the 50-pS K channel activity but also abolished the inhibitory effect of AA and 20-HETE. We conclude that stimulation of adenosine A(?a) receptor increased the 50-pS K channel activity in the TAL, an effect that is achieved by suppression of PLA? activity and 20-HETE-induced inhibition.     

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.     

Activation of adenosine A2A receptors alters postsynaptic currents and depolarizes neurons of the supraoptic nucleus

Supraoptic nucleus (SON) neurons secrete oxytocin or vasopressin in response to various physiological stimuli (e.g., lactation/suckling, dehydration). Released near fenestrated capillaries of the neurohypophysis, these peptides enter the blood and travel to peripheral target organs. The pervasive neuromodulator adenosine, acting at A1 receptors, is an important inhibitory regulator of magnocellular neuroendocrine cell activity. Another high-affinity adenosine receptor exists in this system, however. We examined the physiological effects of adenosine A2A receptor activation and determined its localization among various cell types within the SON. In whole cell patch-clamp recordings from rat brain slices, application of the selective adenosine A2A receptor agonist CGS-21680 caused membrane depolarizations in SON neurons, often leading to increased firing activity. Membrane potential changes were persistent (>10 min) and could be blocked by the selective A2A receptor antagonist ZM-241385, or GDP-beta-S, the latter suggesting postsynaptic sites of action. However, +/--alpha-methyl-(4-carboxyphenyl)glycine or TTX also blocked CGS-21680 effects, indicating secondary actions on postsynaptic neurons. In voltage-clamp mode, application of CGS-21680 caused a slight increase (approximately 8%) in high-frequency clusters of excitatory postsynaptic currents. With the use of specific antibodies, adenosine A2A receptors were immunocytochemically localized to both the magnocellular neurons and astrocytes of the SON. Ecto-5'nucleotidase, an enzyme involved in the metabolism of ATP to adenosine, was also localized to astrocytes of the SON. These results demonstrate that adenosine acting at A2A receptors can enhance the excitability of SON neurons and modulate transmitter release from glutamatergic afferents projecting to the nucleus. We suggest that adenosine A2A receptors may function in neuroendocrine regulation through both direct neuronal mechanisms and via actions involving glia.     

A2A Adenosine Receptors Inhibit ATP-Induced Ca2+ Influx in PC12 Cells by Involving Protein Kinase A

Abstract: The regulatory role of A_2A adenosine receptors in P₂ purinoceptor-mediated calcium signaling was investigated in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with 2- p -(2-carboxyethyl)-phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680), a specific agonist of the A_2A adenosine receptor, the extracellular ATP-evoked rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was inhibited by 20%. Both intracellular calcium release and inositol 1,4,5-trisphosphate production evoked by ATP were not affected by CGS-21680 treatment. However, ATP-evoked Ca²⁺ influx was inhibited following CGS-21680 stimulation. The CGS-21680-mediated inhibition occurred independently of nifedipine-induced inhibition of the [Ca²⁺]_i rise. The CGS-21680-induced inhibition was completely blocked by reactive blue 2. The CGS-21680 effect was mimicked by forskolin and dibutyryl-cyclic AMP and blocked by Rp -adenosine 3',5'-cyclic monophosphothioate, a protein kinase A inhibitor, or by staurosporine, a general kinase inhibitor. The data suggest that in PC12 cells activation of A_2A adenosine receptors leads to inhibition of P₂ purinoceptor-mediated Ca²⁺ influx through ATP-gated cation channels and involves protein kinase A.