Role of adenosine receptor subtypes in rat jejunum in unfed state versus glucose-induced hyperemia

BACKGROUND: Adenosine is a key mediator in intestinal absorptive hyperemia. This study examines the role of adenosine receptor subtypes in the intestinal microvasculature at rest (unfed) and during glucose exposure. MATERIALS AND METHODS: Intravital video microscopy was used to record vascular responses in the rat jejunum in unfed resting states versus active glucose absorption. Two series of experiments were performed: topical adenosine alone and with adenosine receptor antagonists, and topical glucose alone and with adenosine receptor antagonists. RESULTS: We found that distal premucosal arterioles were more reactive to adenosine than were larger inflow arterioles. The selective A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (200 nm), and the A2b receptor antagonist, alloxazine (60 microm), decreased the sensitivity and reactivity of the inflow and premucosal arterioles to adenosine, whereas the selective A2a receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) (200 nm) had no effect on inflow arteriole diameter and only slightly reduced the premucosal arteriolar sensitivity to adenosine. As previously observed, isotonic glucose caused vasodilation (24 +/- 3.4% of the control) in the distal premucosal arterioles. Conversely, premucosal arterioles did not dilate during exposure of the intestine to isotonic mannitol solution that is not actively absorbed. Adenosine A2a RA CSC and A2b RA alloxazine attenuated glucose-induced vasodilation, whereas adenosine A1 RA DPCPX completely abolished glucose-induced dilation. CONCLUSIONS: These findings suggest that resting tone in premucosal vessels appears to be responsive to adenosine mediation rather than inflow arteriolar tone; the adenosine A1, A2a, and A2b receptors all contribute to adenosine-mediated vasodilation in the intestine, with the greatest attenuation seen with A1 receptor antagonism; and other vasoactive mediators might also contribute to glucose-induced jejunal vasodilation, and interaction might exist between adenosine receptors and other mediators.     

ATP- and adenosine-induced relaxation of the smooth muscle of the pig urethra

OBJECTIVES

To investigate relaxation mechanisms for ATP and adenosine in the pig urethra, together with the possible role of ATP in nerve‐evoked urethral relaxations, as ATP is thought to cause bladder smooth muscle contraction via P2X receptors, whereas relaxation is mediated via G‐protein coupled P2Y receptors, and ATP may also induce relaxation via breakdown to adenosine.

MATERIALS AND METHODS

Circular muscle strips from the female pig urethra were mounted in tissue baths to record force; the effects of increasing concentrations of 1–300 µm ATP, the P2‐receptor agonist 2‐methylthioATP (2‐MeSATP), adenosine, the stable adenosine‐analogue, 5′(N‐ethylcarboxamido) adenosine (NECA), ADP, uridine‐triphosphate (UTP) and α,β‐methylene‐ATP were assessed on the spontaneously developed tone. Responses to ATP were further assessed in the presence of G‐protein activator guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS; 1–10 µm ), the G‐protein inhibitor guanosine 5′‐O‐(2‐thio‐diphosphate) (GDPβS; 10–100 µm ), suramin (1–100 µm ), the ecto‐ATPase inhibitor 6‐N,N‐diethyl‐β‐γ‐dibromomethylene‐d ‐adenosine‐5‐triphosphate (ARL 67156, 10–100 µm ), and the suggested P2Y receptor antagonist, reactive blue‐2 (1–100 µm ). The effect of the adenosine (P1) receptor antagonist 8‐(p‐sulphophenyl)theophylline (8‐SPT, 1–100 µm ) on responses to adenosine, and the effects of the adenosine reuptake inhibitor S(p‐nitrobenzyl)‐6‐thioinosine (NBTI, 1–100 µm ) on responses to adenosine and ATP were also assessed. Responses to electrical field stimulation (EFS, 12 and 30 Hz) in the presence of phentolamine (1 µm ), scopolamine (1 µm ) and NΩ‐nitro‐L‐arginine (0.3 mm ) were studied before and after treatment with GTPγS, GDPβS, suramin, reactive blue‐2 and ARL 67156.

RESULTS

Strips were relaxed in a concentration‐dependent manner by exogenously administered ATP and 2‐meSATP, the relaxations being slowly developing and long‐lasting. The relaxant effect evoked by both agonists at 300 µm amounted to about half of the spontaneously developed tone. The relaxation evoked by ATP was not significantly affected by GTPγS, GDPβS, suramin, ARL 67156 or reactive blue‐2. Adenosine induced a concentration‐dependent relaxation of the smooth muscle tone, reaching a maximum of ≈ 70% at 300 µm , whereas 300 µm NECA only relaxed the preparations by ≈ 35%. The adenosine‐induced relaxation was not affected by treatment with 8‐SPT. However, NBTI (1 µm ) significantly reduced the relaxation evoked by 300 µm adenosine. ADP relaxed the smooth muscle tone by ≈ 40% (300 µm ). There was no response to UTP, and the effect of α,β‐methylene‐ATP was negligible (5% relaxation at 100 µm ). EFS caused slowly developing and long‐lasting relaxations that were unaffected by GTPγS, GDPβS, suramin, reactive blue‐2 and ARL 67156.

CONCLUSIONS

These results suggest that exogenous ATP and adenosine relax the smooth muscle of the pig urethra in a manner similar to that evoked by electrical stimulation of nerves, although there was no evidence for involvement of a definable P2Y receptor subtype in these relaxations.

     

Possible role of sildenafil in inhibiting rat vas deferens contractions by influencing the purinergic system

AIM: To evaluate the effect of sildenafil, a selective inhibitor of cyclic guanosine monophosphate (cGMP)-selective type 5 phosphodiesterase, on isolated rat vas deferens and its connections with the purinergic system. METHODS: Epididymal and prostatic portions of isolated vas deferens were placed in organ baths containing Krebs' solution. Contractions were induced by noradrenaline (NA), adenosine triphosphate (ATP), alpha,beta-methylene ATP and electrical field stimulation (EFS). The effect of sildenafil on the contractions was compared with suramin and Evans blue (EB). RESULTS: NA, ATP, alpha,beta-methylene ATP and EFS caused contractions in both portions of vas deferens. NA-induced contractions were unaffected by sildenafil and suramin but potentiated by EB. ATP-induced contractions were non-competitively inhibited in both portions by sildenafil and suramin but potentiated by EB. alpha,beta-methylene ATP-induced contractions were unaffected by sildenafil but were inhibited in both portions by suramin and EB. EFS-induced contractions were inhibited by sildenafil and suramin while potentiated by EB. CONCLUSION: Sildenafil inhibited the contractions in both portions of vas deferens, as did suramin. We have suggested that purinergic system has a role in this antagonism and it seems to be mediated by an ATP-dependent mechanism instead of a receptor interaction.     

Effect of hypothyroidism on the purinergic responses of corpus cavernosal smooth muscle in rabbits

AIMS: Several studies have reported evidence of hormonal abnormalities in 25-35% of impotent men. Hypothyroidism has been reported to occur in 6% of impotent men. In the present study, we examined purinergic relaxation responses in hypothyroidism in an experimental rabbit model and compared them with controls to evaluate the possible involvement of the purinergic pathway. MATERIALS AND METHODS: The study comprised 20 male New Zealand white rabbits. The rabbits were divided into two equal groups. We tested the effects of ATP, alpha beta ATP, and adenosine precontracted with phenylephrine on the isolated corpus cavernosum preparations from control and hypothyroid rabbits. We also evaluated the effects of ATP, alpha beta ATP, and adenosine on the cGMP levels in the isolated corpus cavernosum preparations from control and hypothyroid rabbits. RESULTS: T(3), T(4), and testosterone levels were significantly lower in hypothyroid rabbits. ATP, alpha beta ATP, carbachol, and electrical field stimulation (EFS)-induced frequency-dependent relaxation responses in the isolated rabbit corpus cavernosum strips precontracted with phenylephrine reduced significantly (P < 0.05). Adenosine-induced relaxation responses did not change significantly in hypothyroid rabbits. CONCLUSION: Reduction of relaxation response in hypothyroid rabbits corpus cavernosum can depend on a decreased release of nitric oxide (NO) from nitrergic nerves and endothelium.     

Studies on the mechanisms involved in the ATP-induced relaxation in human and rabbit corpus cavernosum

The effect of ATP in human and rabbit corpus cavernosum (CC) smooth muscle was investigated. Strips of human CC were vertically mounted in an organ bath and the tonic tension was recorded. ATP (0.1-3 mM) induced a concentration-dependent relaxant effect, with a pD2 value of 3.01+/-0.3. The purine-induced relaxation was not affected by L-NAME (100 microM). In rabbit CC, ATP also induced a concentration-dependent relaxation, which was not influenced by L-NAME or by indomethacin (3 microM), with a pD2 value of 3.1 +/-0.4. The ATP-induced relaxant effect in rabbit CC was increased by both the inhibitor of adenosine reuptake, dipyridamole (3 microM) and by the inhibitor of adenosine deaminase, EHNA (0.3 microM). Moreover CGS 15943 (3 microM), an A2a adenosine antagonist, reduced the ATP-induced relaxation. UTP was not able to produce relaxation. The two ATP analogues 2-methylthioATP and alpha,beta-methylene ATP were able to induce relaxation in rabbit CC, with the following order of potency: 2-methylthioATP > ATP > alpha,beta-methylene ATP thus suggesting a role for P2y receptors. However, reactive blue (500 microM), an unspecific P2y antagonist, did not modify the ATP relaxant response. The inhibition of phospholipase C by U73122 (3 microM) and of the endoplasmic reticulum Ca2+ATPase by thapsigargin (1 microM) did not modify the ATP-induced relaxation. The P2x specific antagonist PPADS (30 microM) and suramine (500 microM) were not able to modify the ATP relaxation either in the absence or presence of CGS 15943 (3 microM). These results confirm that ATP acts as a potent and NO-independent relaxant agent of human and rabbit CC. Our findings also show that the ATP effect is partially attributable to the metabolic breakdown of ATP to adenosine, which acts through A2a receptor stimulation, but is also due to a direct stimulation of P2 receptors that are different from the classical P2y and P2X receptor subtypes for ATP.     

The contractile potency of adenosine triphosphate and ecto-adenosine triphosphatase activity in guinea pig detrusor and detrusor from patients with a stable,unstable or obstructed bladder

PURPOSE: We compared the potency of adenosine triphosphate (ATP) and its nonhydrolyzable analogue alpha,beta-methylene ATP for generating contractions in human detrusor smooth muscle from patients with a stable, unstable and obstructed bladders. The different ATP potencies were compared with the ecto-adenosine triphosphatase (ATPase) of these samples. MATERIALS AND METHODS: Contractile experiments were done in vitro by superfusing samples with purines and dose-response curves were generated. Ecto-ATPase activity was measured from the rate of ATP hydrolysis sensitive to the ecto-ATPase inhibitor ARL 67156 with a luciferin-luciferase assay. RESULTS: ATP generated contractions with a mean EC50 of 933 microM. in tissue from stable bladders and was significantly more potent in tissue from unstable and obstructed bladders (EC50 141 and 172 microM., respectively). alpha,beta-methylene ATP was more potent in tissue from stable and unstable bladders (mean combined EC50 3 microM.). In guinea pig detrusor the mean EC50 for ATP and alpha,beta-methylene ATP was 138 and 5.5 microM., respectively. Mean total ATPase activity in unstable bladder biopsies plus or minus standard deviation was about 50% of that in stable bladder biopsies (2.54 +/- 1.50 versus 1.37 +/- 0.46 nmol. per second per mg. protein ). The ARL 67156 sensitive fraction was also significantly less in samples from unstable compared with stable bladders (mean 0.94 +/- 0.41 versus 0.36 +/- 0.26 nmol. per second mg. protein ). CONCLUSIONS: The greater potency of ATP for generating contractions in detrusor from unstable bladders may be due to reduced extracellular hydrolysis, allowing purine greater access to detrusor smooth muscle. This finding may explain atropine resistant purine based contractions in detrusor from unstable bladders.     

Action of adenosine 5'-triphosphate and effects of various purinergic receptor blocking agents on the movements of an isolated guinea pig gallbladder

Exogenously applied adenosine 5'-triphosphate (ATP) caused a biphasic response; a quick relaxation followed by a slow contraction. This response was not affected by atropine, phentolamine, propranolol and tetrodotoxin. Theophylline, quinidine and IBMX reduced ATP-induced contraction and had no effect on ATP-induced relaxation. On the other hand, apamin reduced ATP-induced relaxation and had no effect on ATP-induced contraction. Indomethacin remarkedly reduced ATP-induced contraction without affecting the relaxation. It was suggested that there were two types of receptors (apamin sensitive and apamin resistant) in the guinea-pig gallbladder.     

Adenosine receptor antagonism in acute tacrolimus toxicity.

BACKGROUND: Calcineurin inhibitors induce renal vasoconstriction and oliguria during acute toxicity. We previously demonstrated that the non-specific adenosine receptor antagonist theophylline improved glomerular filtration rate (GFR) and renal blood flow in the setting of acute tacrolimus (TAC) toxicity. This study was undertaken to determine which of the known adenosine receptor subtypes is responsible for the observed effect of theophylline. METHODS: The GFR was measured by clearance of 51Cr-EDTA in anaesthetized, instrumented Sprague-Dawley rats at three time points: at baseline, 60 min after intravenous administration of TAC (0.05 mg/kg) or vehicle (V) and at 100 min after TAC or V. Either DMSO (n = 5) or one of the three available specific adenosine receptor subtype antagonists 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 2 mg/kg, n = 5), a selective A1 receptor antagonist, 8-(3-chlorostyryl) caffeine (CSC, 2 mg/kg, n = 4), a selective A2a receptor antagonist and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-dihydropyridine-3,5 dicarboxylate (MRS1191, 1 mg/kg, n = 5), a selective A3 receptor antagonist, was administered intra-peritoneally prior to the final GFR measurement. Repeated measures analysis of variance was used to detect differences between groups (P < 0.05). RESULTS: Measured GFR declined by 30% from baseline 60 min after TAC. In DMSO treated animals, GFR decreased 51% from baseline at 100 min after TAC, but increased 45% from baseline at 100 min after TAC + MRS1191. CONCLUSIONS: Only administration of the A3 adenosine antagonist increased GFR following TAC, suggesting that this receptor mediates the effect of theophylline on GFR.     

ATP and adenosine act as a mitogen for osteoblast-like cells (MC3T3-E1)

Extracellular ATP, and to a lesser extent adenosine, an ATP metabolite, stimulated cell proliferation in osteoblast-like cells (MC3T3-E1). ATP increased cytosolic Ca²⁺ due to Ca²⁺ mobilization from intracellular storage in the same concentration range of the nucleotide as that effective for DNA synthesis, suggesting the mediation of the phospholipase C/Ca²⁺ system in the mitogenic action. Since adenosine induced no Ca²⁺ mobilization, P₂-purinergic receptor appears to be associated with ATP actions. The growth-promoting effect of ATP was not inhibited by H7, a protein kinase C inhibitor, and indomethacin, a cyclooxygenase inhibitor, indicating no involvement of activation of protein kinase C and production of prostaglandins in ATP-induced mitogenic signals. Either ATP or adenosine remarkably and synergistically potentiated platelet derived growth factor-induced DNA synthesis. These findings suggest that extracellular ATP and adenosine may play a physiological role in the regulation of bone formation. Received: 17 January 1995 / Accepted: 21 March 1995     

Effect of chronic renal failure on the purinergic responses of corpus cavernosal smooth muscle in rabbits

OBJECTIVE: To examine purinergic relaxation responses in chronic renal failure (CRF) in an experimental rabbit model, and thus evaluate the possible involvement of the purinergic system in erectile dysfunction with CRF. MATERIALS AND METHODS: The relaxant effects of ATP were measured in strips of corpus cavernosum smooth muscle taken from control and CRF rabbits. CRF was induced in New Zealand white rabbits as previously described. Penises were excised from CRF rabbits 4 weeks after inducing uraemia. In an organ bath the strips from controls and CRF rabbit corpus cavernosum were pre-contracted with phenylephrine and increasing doses of adenosine and ATP added. RESULTS: In the pre-contracted rabbit cavernosal tissue the relaxations induced by adenosine and ATP were unchanged in CRF. CONCLUSION: The lack of any relaxant effect of adenosine or ATP on the relaxation of cavernosal smooth muscle in rabbits with CRF might be because the relaxant effects of these agents are endothelium-independent and the endothelial purinergic receptor density was unchanged in CRF.     

Intracellular mechanism of mitochondrial adenosine triphosphate-sensitive potassium channel activation with isoflurane

The precise mechanism of isoflurane and mitochondrial adenosine triphosphate-sensitive potassium channel (mitoK(ATP)) interaction is still unclear, although the mitoK(ATP) is involved in isoflurane-induced preconditioning. We examined the role of various intracellular signaling systems in mitoK(ATP) activation with isoflurane. Mitochondrial flavoprotein fluorescence (MFF) was measured to quantify mitoK(ATP) activity in guinea pig cardiomyocytes. To confirm isoflurane-induced MFF, cells were exposed to Tyrode's solution containing either isoflurane (1.0 +/- 0.1 mM) or diazoxide and then both drugs together (n = 10 each). In other studies, the following drugs were each added during isoflurane administration: adenosine or the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (SPT); the protein kinase C (PKC) activators phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu); the PKC inhibitors polymyxin B and staurosporine; the tyrosine kinase inhibitor lavendustin A; or the mitogen-activated protein kinase inhibitor SB203580 (n = 10 each). Isoflurane potentiated MFF induced by diazoxide (100 micro M), and diazoxide also increased isoflurane-induced MFF. PMA (0.2 micro M), PDBu (1 micro M), and adenosine (100 micro M) induced MFF. However, SPT (100 micro M), polymyxin B (50 micro M), staurosporine (200 nM), lavendustin A (0.5 micro M), and SB203580 (10 micro M) all failed to inhibit the effect of isoflurane. Our results show that isoflurane, adenosine, and PKC activate mitoK(ATP). However, our data do not support an action of isoflurane through pathways involving adenosine, PKC, tyrosine kinase, or mitogen-activated protein kinase. These results suggest that isoflurane may directly activate mitoK(ATP). IMPLICATIONS: Our results show that isoflurane activates mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels, but not through pathways involving adenosine, protein kinase C, tyrosine kinase, or p38 mitogen-activated protein kinase. Isoflurane may directly activate mitoK(ATP) channels.     

Adenosine responses in experimental vein bypass grafts

Purpose: Veins develop unique endothelial and smooth muscle cell physiologic phenotypes after implantation as vein grafts in the arterial circulation. Receptor-mediated relaxation is reduced or absent in these grafts. This study examines the responses of vein grafts to adenosine, a known potent endogenous vasodilator, and compares these responses with the native veins and arteries. Methods: The presence of adenosine receptors (A₁ and A₂) by radioligand binding and the in vitro responses to the adenosine analogue N-ethyl-carboxyamido-adenosine were assessed in precontracted common carotid jugular vein bypass grafts placed in New Zealand white rabbits for 28 days. Results were compared with those obtained in precontracted jugular veins and carotid arteries. Both endothelialized and de-endothelialized vessels were studied. The contribution of nitric oxide (NO) and prostanoid production to relaxation was also determined by preincubation with the specific inhibitors <!--**Check here**-->l-monomethylarginine and indomethacin, respectively. Finally, the in vitro relaxation in response to the respective A₁ and A₂ adenosine receptor agonists R-phenyl-isopropyl-adenosine and CGS-21680 was also examined. Results: The results show that the adenosine-induced responses of the vein grafts differ from those of the jugular vein and carotid artery. First, in contrast to the carotid artery, vein graft adenosine-mediated relaxation is NO and prostanoid dependent, similar to the response of the jugular vein. Second, A₁ receptor activation in the vein graft produces an endothelium-dependent contractile response. Third, the A₂ receptor-mediated responses in the vein grafts appear to be independent of the endothelium. Fourth, radioligand studies show the presence of both receptor subtypes (A₁ and A₂) on the vein grafts with a ratio (A₁/A₂ = 1.4) closer to that of the jugular vein (A₁/A₂ = 1.8) than to that seen in the carotid artery (A₁/A₂ = 0.5). Conclusions: Vein graft adenosine responses appear to be unique in that they neither maintain a venous phenotype nor acquire an arterial phenotype. In particular, endothelial A₁ receptor-mediated responses change from relaxation to contraction, and receptor activated NO-mediated relaxation is preserved within the vein grafts probably via A₂ receptor signalling. (J Vasc Surg 1998;28:929-38.)     

Vagal involvement in the action of exogenous adenosine triphosphate on reflex renal sympathetic nerve activity

The reason why adenine compounds when used as hypotensive agents are devoid of significant reflex sympathetic activity, such as rebound hypertension and tachycardia, is not clearly understood. This study, performed on alpha-chloralose-anesthetized dogs, examined, first, the effects of adenosine triphosphate (ATP) and adenosine as compared with those of sodium nitroprusside on efferent renal sympathetic nerve activity (RSNA), as an indicator of general reflex sympathetic activity, and second, whether vagal involvement could be demonstrated in the action of ATP and adenosine on RSNA. Renal sympathetic nerve activity increased progressively with increasing doses of sodium nitroprusside (5, 10, and 20 micrograms/kg) and adenosine (0.5, 2.0, and 4.0 mg/kg), whereas ATP suppressed RSNA at 2.0 and 4.0 mg/kg. High doses of ATP and adenosine (4.0 mg/kg) were injected into intact (n = 7) and vagotomized dogs (n = 7). Both ATP and adenosine induced rapid onset of hypotension without rebound hypertension and tachycardia. After vagotomy, the attenuation of RSNA by ATP was completely abolished and rebound hypertension and tachycardia were observed. Vagotomy did not alter the effect of adenosine on RSNA. It is concluded that ATP-induced hypotension is associated with attenuation of sympathetic efferent nerve activity mediated through vagal afferent pathways. Vagal afferent impulses are thought to be one of the mechanisms that inhibit reflex sympathetic activities, such as rebound hypertension after ATP-induced hypotension. The mechanisms by which adenosine inhibits reflex sympathetic activity are not, however, secondary to vagal afferent involvement and must be multifactorial.     

A2B adenosine receptor-mediated induction of IL-6 promotes CKD

Chronic elevation of adenosine, which occurs in the setting of repeated or prolonged tissue injury, can exacerbate cellular dysfunction, suggesting that it may contribute to the pathogenesis of CKD. Here, mice with chronically elevated levels of adenosine, resulting from a deficiency in adenosine deaminase (ADA), developed renal dysfunction and fibrosis. Both the administration of polyethylene glycol-modified ADA to reduce adenosine levels and the inhibition of the A(2B) adenosine receptor (A(2B)R) attenuated renal fibrosis and dysfunction. Furthermore, activation of A(2B)R promoted renal fibrosis in both mice infused with angiotensin II (Ang II) and mice subjected to unilateral ureteral obstruction (UUO). These three mouse models shared a similar profile of profibrotic gene expression in kidney tissue, suggesting that they share similar signaling pathways that lead to renal fibrosis. Finally, both genetic and pharmacologic approaches showed that the inflammatory cytokine IL-6 mediates adenosine-induced renal fibrosis downstream of A(2B)R. Taken together, these data suggest that A(2B)R-mediated induction of IL-6 contributes to renal fibrogenesis and shows potential therapeutic targets for CKD.     

Ischemia postconditioning protects dermal microvascular endothelial cells of rabbit epigastric skin flaps against apoptosis via adenosine A2a receptors

Background: It has been shown that endogenous adenosine-induced by ischemia postconditioning attenuates apoptosis in recent studies; however, they focus only on parenchymal cells. The detailed mechanism has not been clearly clarified in any research and the subtype of adenosine receptors involved remains unknown. In our study, dermal microvascular endothelial cells (DMECs) are used to explore the role of adenosine A_2a receptor in the anti-apoptotic effects of ischemic postconditioning.

Material and methods: The epigastric skin flaps of rabbits were elevated. After 4?h of ischemia, the flaps were either abruptly reperfused or postconditioned by six cycles of brief reperfusion (15s) and re-ischemia (15s). Adenosine A2a receptor agonist (CGS-21680) and antagonist (ZM-241385) were used separately in other groups. The apoptosis-related proteins and adenosine A2a receptors were determined by immunohistochemical staining. Then apoptosis index was calculated by TUNEL.

Results: Ischemia/reperfusion caused severe damages in DMECs of flaps as demonstrated by an increase in apoptosis index and an increase in expressions of apoptosis-related proteins, which can be significantly attenuated by IPC treatment or exposure to a selective adenosine A_2a receptor agonist (all p values <.05). Meanwhile, the anti-apoptosis effects of IPC can be blocked by a selective adenosine A_2a receptor antagonist. Statistical analysis revealed that the increase of apoptosis index closely correlated inversely with the relative increase of adenosine A_2a receptors (p?Conclusions: Ischemia postconditioning protects DMECs of rabbit skin flap against apoptosis via activation of adenosine A_2a receptors.     

The effects of adenosine A2B receptor inhibition on VEGF and nitric oxide axis-mediated renal function in diabetic nephropathy

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease worldwide. The pathophysiologic mechanisms of diabetic nephropathy are incompletely understood but include overproduction of various growth factors and cytokines. Upregulation of vascular endothelial growth factor (VEGF) is a pathogenic event occurring in most forms of podocytopathy; however, the mechanisms that regulate this growth factor induction are not clearly identified. A_2B receptors have been found to regulate VEGF expression under hypoxic environment in different tissues. One proposed hypothesis in mediating diabetic nephropathy is the modulation of VEGF-NO balance in renal tissue. We determined the role of adenosine A_2B receptor in mediating VEGF overproduction and nitrite in diabetic nephropathy. The renal content of A_2B receptors and VEGF was increased after 8 weeks of diabetes induction. The renal and plasma nitrite levels were also reduced in these animals. In vivo administration of A_2B adenosine receptor antagonist (MRS1754) inhibited the renal over expression of VEGF and adverse renal function parameters. The antagonist administration also improved the kidney tissue nitrite levels. In conclusion, we demonstrated that VEGF induction via adenosine signaling might be the critical event in regulating VEGF-NO axis in diabetic nephropathy.     

Preconditioning and adenosine protect human proximal tubule cells in an in vitro model of ischemic injury

Renal ischemic reperfusion injury results in unacceptably high mortality and morbidity during the perioperative period. It has been recently demonstrated that ischemic preconditioning or adenosine receptor modulations attenuate renal ischemic reperfusion injury in vivo. An in vitro model of ischemic renal injury was used in cultured human proximal tubule (HK-2) cells to further elucidate the protective signaling cascades against renal ischemic reperfusion injury. ATP depletion preconditioning (1 h of antimycin A and 2-deoxyglucose treatment followed by 1 h of recovery), adenosine, an A(1) adenosine receptor selective agonist, or an A(2a) adenosine receptor selective agonist significantly attenuated subsequent severe ATP depletion injury of HK-2 cells. In contrast, an adenosine receptor antagonist failed to prevent protection induced by ATP depletion preconditioning. Cytoprotection by ATP depletion preconditioning or A(1) adenosine receptor activation was prevented by inhibitors of extracellular signal-regulated mitogen-activated kinases, protein kinase C, and tyrosine kinases. The A(1) and A(2a) adenosine receptor-mediated cytoprotection were also dependent on G(i/o) proteins and PKA activation, respectively. It is concluded that ATP depletion preconditioning and A(1) and A(2a) adenosine receptor activation protect HK-2 cells against severe ATP depletion injury via distinct signaling pathways.     

Endogenously produced adenosine regulates articular cartilage matrix homeostasis: enzymatic depletion of adenosine stimulates matrix degradation

OBJECTIVE: Enhanced extracellular levels of adenosine have been shown to inhibit experimentally induced cartilage degradation. The objective of this study was to investigate the role of adenosine and A(2)adenosine receptors in regulating cartilage homeostasis in the absence of inflammatory stimuli. METHODS: Cartilage explants were exposed to adenosine deaminase (ADA) to deplete extracellular adenosine, and conditioned medium was collected for evaluation of glycosaminoglycan (GAG), prostaglandin E(2)(PGE(2)), nitric oxide (NO), and matrix metalloproteinases-3 and -13 (MMP-3, MMP-13) levels. In a second set of experiments, cartilage incubated with ADA was simultaneously exposed to the adenosine kinase inhibitor 5'-iodotubercidin (ITU) to inhibit adenosine breakdown, or to the A(2A)adenosine receptor agonist N(6)-[2-(3,5-dimethoxyphenyl)-ethyl]adenosine (DPMA). Finally, explants were incubated with the adenosine receptor antagonists ZM241385, CGS15943, theophylline or caffeine to block normal receptor activation by endogenous adenosine. RESULTS: Exposure to ADA induced a concentration-dependent increase in GAG release and production of total MMP-3, MMP-13, PGE(2), and NO. Both ITU and DPMA inhibited the ADA-mediated increases in GAG release and PGE(2), and NO production, but only ITU inhibited MMP-13 release. Exposure to ZM 241385 increased GAG, MMP-3 and MMP-13 release. Additionally, CGS 15943 increased MMP-3 production while theophylline increased GAG, PGE(2), and NO release. CONCLUSIONS: Endogenous adenosine levels appear to regulate cartilage matrix homeostasis even in the absence of inflammation. Regulation occurs, at least in part, through activation of cell surface receptors. This study suggests that autocrine and paracrine responses to adenosine release are important for maintenance of healthy articular cartilage.     

Role of spinal adenosine receptors in modulating the hyperesthesia produced by spinal glycine receptor antagonism

The intrathecal administration of strychnine in rats yields a prominent touch-evoked allodynia. The effects of an intrathecally administered A1 adenosine agonist: N6-(L-2-phenylisopropyl)-adenosine (LPIA) or an A2 adenosine agonist: 5'-(N-ethyl carboxamido)-adenosine (NECA), on this touch-evoked hyperesthesia were examined. Over the range of 0.3-1.0 nmol these agents produced a dose-dependent inhibition of the strychnine-evoked hyperesthesia. This inhibition was reversed following intraperitoneal injection of caffeine, an adenosine receptor antagonist. No statistical differences between LPIA and NECA were recorded. The powerful effect of adenosine analogues on strychnine hyperesthesia occur at doses that have only a mild analgesic effect on the thermally evoked hot-plate response. This effect is in contrast to opioids, which have been reported to be only minimally effective against strychnine-evoked hyperesthesia. The characteristics of the strychnine hyperesthesia appear to mimic the clinical phenomenon observed in patients suffering from sensory dysesthesia following nerve injury and suggest a possible role for the adenosine receptor in certain pain states.     

Cyclosporine-induced adenosine triphosphate depletion in murine T and B lymphocytes

Adenosine metabolism in C57BL/6 mouse spleen cells was studied. Adenosine triphosphate (ATP) levels in resting T cells were 26.9 +/- 3.4 ng/10(5) cells compared with 16.5 +/- 3.1 ng/10(5) cells in resting B cells. Cyclosporine (CSA) caused a prompt and severe ATP depletion in both T and B cells, which could be mitigated by the addition of adenosine. B cell ATP levels were returned to normal while T cell levels were only partially restored. The adenosine deaminase inhibitor erythro-9-(2 hydroxy-3 nonyl) adenine (EHNA) also caused ATP depletion in T and B cells, which could similarly be prevented in part by the addition of adenosine. However, when CSA and EHNA were combined, adenosine could no longer protect ATP pools and severe ATP depletion in T and B cells occurred. This suggests that CSA and EHNA affect different steps in the conversion of adenosine to ATP. Although both T and B cell ATP levels were affected by CSA, the ability of supplementary substrate to restore ATP levels to normal in B cells but not in T cells may explain the apparent selective effect of CSA impairing T cell functions with sparing of B cell functions. Furthermore, if causing ATP depletion is associated with immunosuppressive activity, EHNA may be useful in potentiating the immunosuppressive effects of CSA.