Evidence for adenylate cyclase-coupled A1-adenosine receptors on ventricular cardiomyocytes from adult rat and dog heart

Isolated metabolically stable cardiomyocytes from adult rats and mongrel dogs were used to characterize the mechanism underlying the antiadrenergic effect of adenosine. In a system not affected by cellular heterogeneity, isoproterenol (3 x 10(-9) M - 10(-5) M) in the presence of adenosine deaminase (5U/ml) dose dependently increased cellular cAMP (5-80 pmol/mg). The effect of isoproterenol (0.1 microM) was inhibited by various adenosine derivatives, the rank order of potency being in the rat: (-)-N6-(R-phenyl-isopropyl)-adenosine (R-PIA) greater than 5'-N-ethylcarboxamidoadenosine (NECA) greater than S-PIA, and in the dog NECA greater than R-PIA greater than S-PIA. The cAMP increase induced by forskolin (1 microM) was attenuated in the rat by R-PIA. 8-phenyltheophylline (3 microM) antagonized the effect of R-PIA on isoproterenol-stimulated cAMP formation. Basal cAMP content was not influenced by R-PIA or NECA. Omission of adenosine deaminase from the incubation medium attenuated the isoproterenol-induced cAMP increase in the rat by about 30%. Our findings provide evidence for the presence of adenylate cyclase-coupled A1-adenosine receptors on cardiomyocytes which may mediate the antiadrenergic effect of adenosine in the heart.     

Identification of Adenosine Receptor in Rat Pineal Gland: Evidence for A-2 Selectivity

We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyladenosine [( 3H]CHA), R-N6-[3H]phenylisopropyladenosine [( 3H]R-PIA), and 5'-N-ethylcarboxamido[3H]adenosine [( 3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A-1-selective ligands (CHA and R-PIA) had less than or equal to 10% specific binding. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 nM) or R-PIA (50 microM). To characterize this binding, we used the N-ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 +/- 10 nM and an apparent Bmax of 1,060 +/- 239 fmol/mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM) (-25%), a result suggesting the involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA greater than 2-chloroadenosine greater than S-adenosyl-L-homocysteine greater than CHA. Binding was also displaced by 3-isobutyl-1-methylxanthine (IC50 = 23.6 microM). These findings are consistent with the selective labeling by [3H]NECA of an A-2-type adenosine receptor in rat pineal membranes.     

Evidence for the presence of A1 and A2 adenosine receptors in the ventral aorta of the dogfish shark,Squalus acanthias

Isolated, endothelium-free rings of vascular smooth muscle (VSM) from the ventral aorta of the dogfish shark, Squalus acanthias, were used to examine the vasoactive effects of various adenosine agonists. Cumulative addition of 2-chloroadenosine (2 Cl-ADO) over the concentration range 10 nM-1 mM resulted in a biphasic response, with a significant increase in tension at 1 microM and a more significant decline in tension at 100 microM and 1 mM, suggesting that this tissue may possess both A1 and A2 adenosine receptors. N6-Cyclopentyladenosine (N-6 CPA) and N6-(2-phenylisopropyl)adenosine, R(-)isomer (R-PIA), generally considered to be more A1 specific, also produced slight, but significant increases in tension, but only at relatively high concentrations. The more specific A1 agonist, N6-(25)-[2-endo-norbonyl] adenosine [(S)-ENBA] produced a significant increase in tension at 1 pM, reaching 28% above control at 10 nM. The response to (S)-ENBA was also biphasic, with a fall in tension at 10 microM. The relatively non-specific agonist 5'-N-ethylcarboxamidoadenosine (NECA) produced a small, but significant, increase in tension at 1 microM, with no subsequent decline in tension at higher concentrations. These results allow us to assign a tentative structure-activity relationship (SAR) for an increase in tension of (S)-ENBA much much greater than R-PIA greater than or equal to 2-Cl ADO = N-6 CPA = NECA; for the decrease, the SAR is (S)-ENBA greater than 2-Cl ADO greater than R-PIA greater than N-6 CPA = NECA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human basophil/mast cell releasability. VII. Heterogeneity of the effect of adenosine on mediator secretion

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than N6-(R-phenyl-isopropyl)-adenosine (R-PIA) inhibited in vitro anti-IgE-induced histamine and peptide leukotriene C4 (LTC4) release from human basophils in a concentration-dependent fashion. Micromolar concentrations of adenosine, NECA and R-PIA potentiated the anti-IgE-stimulated release of histamine and LTC4 from human lung parenchymal mast cells. Submillimolar concentrations of adenosine, NECA and R-PIA inhibited in a concentration dependent manner the release of histamine and prostaglandin D2 (PGD2) from skin mast cells challenged with anti-IgE. These results demonstrate marked heterogeneity of the modulatory effect exerted by adenosine on mediator release from human basophils and mast cells.     

Regulation of bovine sperm motility and cyclic adenosine 3',5'-monophosphate by adenosine and its analogues

Cyclic adenosine 3',5'-monophosphate (cAMP) is known to mediate mammalian sperm function. Progress in understanding the mechanism of the control of cAMP levels in mammalian sperm has been hampered, however, by an inability to identify a physiological regulator of adenylate cyclase. In this report we provide evidence that adenosine, 2'-deoxyadenosine, and a number of other adenosine analogues that activate adenylate cyclase in other tissues stimulate bovine caudal sperm motility, and we suggest that they do so through elevation of cAMP levels. We have demonstrated that these compounds elevate cAMP levels in and stimulate the motility of mature bovine caudal sperm in the same concentration range (20-300 microM). In addition, we report that these same nucleosides, under appropriate conditions, elevate cAMP levels and initiate motility in immature caput sperm. Adenosine analogue structure-activity relationships carried out with caudal sperm indicate that substitution at position 2 in the purine ring in the adenosine molecule leads to enhanced activity, while substitution at the N-6 amino group reduces potency. Nucleosides that do not stimulate motility in caudal sperm do not elevate cAMP levels. We postulate that adenosine is a physiological regulator of sperm motility and suggest that it and its analogues owe their action to elevation of cAMP levels.     

Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex.

Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.     

Evidence for the involvement of calmodulin in mouse sperm capacitation

Although Ca(2+) is of fundamental importance in mammalian sperm capacitation, its downstream targets have not been definitively demonstrated. The purpose of this study was to use the calmodulin (CaM) antagonists W7 and calmidazolium (CZ) to investigate the possible role of CaM, a Ca(2+)-specific binding protein, in capacitation. Sperm membrane changes associated with capacitation were assessed by the B pattern after chlortetracycline staining and by the ability to undergo the acrosome reaction (AR) in response to lysophosphatidylcholine (LPC). The percentage of B pattern sperm was significantly inhibited by W7 or CZ in a concentration-dependent manner. At 100 microM W7 or 10 microM CZ, these inhibitors also significantly reduced the sperm's ability to undergo the LPC-induced AR. Inhibition of the B pattern and the LPC-induced AR was overcome by exogenous cAMP analogues. Treatment of the sperm with 100 microM W7 also resulted in a significant decrease in their ability to fertilize eggs in vitro. At 100 microM, W5, a less potent dechlorinated W7 analogue, had no effect on the B pattern, LPC-induced AR, or fertilization competence. Sperm viability and protein tyrosine phosphorylation were not substantially affected by 100 microM W7 (relative to 100 microM W5) or 10 microM CZ; however, the percentages of motile and hyperactivated sperm were significantly reduced. The antagonist-inhibited sperm motility was restored by dilution in control medium, but not by cAMP analogues. These results suggest that CaM participates in the regulation of membrane changes important for mouse sperm capacitation, at a point upstream from cAMP, and that this pathway is at least partially separable from pathways controlling tyrosine phosphorylation and hyperactivation.     

Inhibition of adenosine-metabolizing enzymes modulates mouse sperm fertilizing ability: A changing role for endogenously generated adenosine during capacitation

The effect of inhibiting adenosine-metabolizing enzymes on sperm fertilizing ability was studied to investigate a possible role for endogenously generated adenosine in the regulation of capacitation. The compounds used have been shown to be effective inhibitors of the relevant enzymes in similarly incubated mouse sperm suspensions. Inhibition of 5′-nucleotidase activity with α,β-methylene adenosine 5′-diphosphate (AMPCP), to reduce available endogenous adenosine, caused a dose-dependent inhibition of the fertilizing ability of partially capacitated spermatozoa, which was significant with 100 and 250 μM AMPCP. Conversely, inhibition of adenosine deaminase with 100 nM coformycin, to increase available endogenous adenosine, promoted the fertilizing ability of partially capacitated spermatozoa when the fertilization rate of control suspensions was low. However, coformycin had no effect on sperm suspensions with moderate fertilizing ability, and it inhibited fertilizing ability when added to capacitated spermatozoa. These data are consistent with a promotion of the early stages of capacitation by endogenously generated adenosine and suggest that sensitivity to adenosine changes as capacitation proceeds. Because the majority of adenosine-metabolizing enzyme activity resides in or is directed toward the extracellular compartment in such suspensions, these effects of adenosine may be mediated at the outer surface of the cell. By interacting with receptors on adenylate cyclase, externally produced adenosine could modulate intracellular levels of cyclic adenosine monophosphate (cAMP), thereby influencing fertilizing ability.     

Adenosine and catecholamine agonists speed the flagellar beat of mammalian sperm by a non-receptor-mediated mechanism

Activation of rapid motility apparently is one of the first steps of sperm capacitation and can be studied in vitro. Previously we found that 2-chloro-2'-deoxyadenosine or the catecholamine isoproterenol activates mouse sperm motility in vitro via a pathway mediated by cAMP that requires extracellular Ca2+, the atypical sperm adenylyl cyclase, and sperm-specific protein kinase A. We now show that several other adenosine analogs and catecholamines accelerate the flagellar beat of mouse and human sperm. Unexpectedly, the potent adenosine receptor agonist CGS21680 does not accelerate the beat, and the adenosine receptor antagonist DPCPX does not diminish the accelerating action of 2-chloro-2'-deoxyadenosine. The pharmacological profile for activation by catecholamines is also unusual. Both agonists and antagonists of beta-adrenergic receptors elevate the beat frequency. Moreover, both l-(-) and d-+ isomers of epinephrine, norepinephrine, and isoproterenol produce similar acceleration of the beat. In contrast, inhibitors of equilibrative nucleoside transporters effectively slow the onset of the accelerating action of adenosine analogs. Replacement of external Na+ with Li+ also diminishes the accumulation of cAMP and slows the resultant accelerating action of 2-chloro-2'-deoxyadenosine, suggesting the involvement of a Na+-dependent concentrative nucleoside transporter. Our results show that adenosine and catecholamine agonists act in a novel signaling pathway that does not involve G protein-coupled cell-surface receptors that link to conventional adenylyl cyclases. Instead, adenosine and analogs may be transported into sperm via equilibrative and concentrative nucleoside transporters to act on unknown intracellular targets.     

Implication of cAMP during porcine sperm capacitation and protein tyrosine phosphorylation

Second messengers are involved in sperm fertilizing potential, as both motility and the acrosome reaction are influenced by cAMP. Moreover, the activity of cyclic nucleotides is implicated in the appearance of tyrosine phosphorylated sperm proteins, which is associated with capacitation in the mammalian spermatozoa. Nevertheless, the involvement of the cAMP/protein kinase A (PK-A) pathway during pig sperm capacitation may be different from that observed in other mammals. The objective of the present study was to clarify the cAMP/PK-A pathway during the capacitation of porcine spermatozoa and to evaluate this impact on the p32 sperm tyrosine phosphoprotein appearance. The presence of p32 was assessed after incubating fresh pig sperm with IBMX/db-cAMP, H-89, a PK-A inhibitor or bistyrphostin, a tyrosine kinase inhibitor, in capacitating (CM) or non-capacitating conditions (NCM) by immunoblotting SDS-extracted and separated sperm proteins using an anti-phosphotyrosine antibody. When pig spermatozoa were incubated in CM supplemented with H-89 (50 microM) or bistyrphostin (1.2 microM), capacitation decreased significantly (P < 0.001). The p32 sperm tyrosine phosphoprotein, previously shown to be associated with capacitation of porcine sperm though not necessarily an end point of this phenomenon, was not modulated by IBMX/db-cAMP (100 microM/1 mM), H-89 (50 microM) nor bistyrphostin (1.2 microM). Our results indicate, therefore, that pig sperm are regulated somewhat differently than as described for other mammals, because although the cAMP/PK-A and tyrosine kinase pathways are involved in capacitation, they do not influence the appearance of p32.     

Stimulation of glycogenolysis and vasoconstriction by adenosine and adenosine analogues in the perfused rat liver.

Infusion of adenosine into perfused rat livers resulted in transient increases in glucose output, portal-vein pressure, the effluent perfusate [lactate]/[pyruvate] ratio, and O2 consumption. 8-Phenyltheophylline (10 microM) inhibited adenosine responses, whereas dipyridamole (50 microM) potentiated the vasoconstrictive effect of adenosine. The order of potency for adenosine analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) greater than L-phenylisopropyladenosine greater than cyclohexyladenosine greater than D-phenylisopropyladenosine greater than 2-chloroadenosine greater than adenosine, consistent with adenosine actions modulated through P1-purine receptors of the A2-subtype. Hepatic responses exhibited homologous desensitization in response to repeated infusion of adenosine. Adenosine effects on the liver were attenuated at lower perfusate Ca2+ concentrations. Indomethacin decreased hepatic responses to both adenosine and NECA. Whereas adenosine stimulated glycogen phosphorylase activity in isolated hepatocytes, NECA caused no effect in hepatocytes. The response to adenosine in hepatocytes was inhibited by dipyridamole (50 microM), but not 8-phenyltheophylline (10 microM). The present study indicates that, although adenosine has direct effects on parenchymal cells, indirect effects of adenosine, mediated through the A2-purinergic receptors on another hepatic cell type, appear to play a role in the perfused liver.     

Adenosine receptors, cyclic AMP accumulation, and DNA-synthesis in aortic smooth muscle cell cultures of adult and neonatal rats.

The effects of two adenosine analogs on cyclic AMP (cAMP) accumulation and DNA synthesis were studied in cultured smooth muscle cells (SMCs) isolated from adult and neonatal rat arteries. N-ethylcarboxamido adenosine (NECA) dose-dependently increased intracellular cAMP levels and appeared to be more potent in adult than in neonatal SMCs. R-phenylisopropyl adenosine (R-PIA), in nanomolar concentrations, counteracted the increase in cAMP evoked by 10 microM forskolin in adult but not in neonatal SMCs, indicating that the enhanced "A2" response seen in adult SMCs was not due to a lack of "A1" receptors in these cultures. Binding experiments performed using the adenosine antagonist XAC did not reveal any differences in the number or affinity of the adenosine receptors between neonatal and adult SMCs. This indicates effects presumably on the G-protein level. A high capacity to spontaneously synthesize DNA and a weak response to platelet-derived growth factor (PDGF) were seen in the neonatal SMCs. Furthermore, NECA had no effect on PDGF-induced DNA synthesis in these cells. In contrast, adult SMCs presented a low rate of spontaneous DNA synthesis and a marked proliferative response to PDGF, which was inhibited by NECA. This inhibition paralleled the increase in cAMP elicited by NECA. Our findings suggest that neonatal and adult SMCs differ both in their response to growth factors and growth inhibitors.     

Phosphodiesterase activity of mouse sperm incubated under conditions that modulate fertilizing potential in vitro

Cyclic AMP has been implicated as a regulator of capacitation, but the control of its metabolism in sperm remains obscure. A recent study of mouse sperm has shown capacitation-related changes in the activities of both adenylate cyclase, which increased during incubation, and cyclic nucleotide phosphodiesterase, which decreased. The present study was conducted to extend these observations by measuring phosphodiesterase activity in sperm incubated in media with modified calcium and/or glucose content, conditions known to modulate fertilizing ability. Phosphodiesterase activity of sequential sperm samples, taken first when sperm are essentially uncapacitated and then when they are either partially or completely capacitated, decreased with time under all conditions, and in each case the greater fall in activity was seen in the medium that would support the greater change in fertilizing ability of the sperm population. Sperm washed by centrifugation to remove epididymal fluid also displayed a reduction in phosphodiesterase activity with time. The medium surrounding the sperm contained about half of the total phosphodiesterase activity, as well as 5′-nucleotidase and adenosine deaminase. The crude enzyme preparation showed complex kinetic behavior when assayed over a range of cAMP concentrations, but the reduction in activity with time was seen at all substrate levels. The observed changes in phosphodiesterase activity, together with the increased adenylate cyclase activity seen under these sperm incubation conditions, would increase cAMP availability with time, thus providing further evidence for a fundamental role for cAMP in controlling the events of capacitation.     

A possible mechanism of action for fertilization promoting peptide,a TRH-related tripeptide that promotes capacitation and fertilizing ability in mammalian spermatozoa

Fertilization promoting peptide (FPP), a tripeptide structurally related to thyrotrophin releasing hormone (TRH), has been shown to stimulate capacitation and fertilizing ability in both mouse and human spermatozoa, but the mechanisms of action involved in these responses are currently unknown. In the present study utilizing epididymal mouse spermatozoa, we have compared the ability of FPP, TRH, and pyroglutamylphenylalanineprolineamide (an uncharged structurally related tripeptide found in seminal plasma) to stimulate capacitation. At 50 nM, the mean concentration of FPP found in human seminal plasma, only FPP produced a significant response. This suggests that if a receptor is involved, it is one distinct from the TRH receptor. A significant response to FPP required the presence of extracellular Ca²⁺, with 90 μm Ca²⁺ being sufficient to support a stimulation of capacitation. The addition of FPP to suspensions at later stages of capacitation indicated that the nature of the response changed, such that addition of FPP to capacitated suspensions inhibited spontaneous acrosome reactions; however, FPP-treated, cells were still able to undergo acrosomal exocytosis in response to progesterone, a physiological agonist of acrosomal exocytosis. Because earlier studies had identified a similar capacitation-related change in response to adenosine, being stimulatory early in capacitation and inhibitory later in capacitation, we investigated the possibility that FPP and adenosine might be acting via the same pathway. The combination of FPP plus adenosine, whether used at low, non-stimulatory concentrations or high, maximally-stimulatory concentrations, was more effective in promoting capacitation than either compound used individually. As observed with FPP, addition of adenosine to capacitated cells inhibited spontaneous acrosome loss but did not inhibit exocytosis in response to progesterone. This suggests that the two molecules are affecting a common pathway. Since adenosine, acting via specific cell surface receptors, can stimulate fertilizing ability and adenylate cyclase activity in uncapacitated cells and then inhibit enzyme activity in capacitated cells, we propose that FPP may act by modulating the adenylate cyclase/cyclic AMP signal transduction pathway. In vivo, FPP, which would contact spermatozoa at ejaculation and probably remain bound to cells for some time, could stimulate capacitation as the spermatozoa ascend the female tract; adenosine, present in seminal plasma and the female tract, could either augment FPP's action or replace it if FPP is lost from the cell surface. We therefore suggest that FPP and adenosine, by modulating adenylate cyclase activity to promote capacitation but inhibit spontaneous acrosomal exocytosis, may provide an endogenous mechanism that helps to optimize the fertilizing potential of the few sperm cells that reach the site of fertilization in vivo. © 1996 Wiley-Liss, Inc.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Adenosine; a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on human neutrophils

Adenosine specifically inhibits superoxide anion generation by N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils without affecting either degranulation or "aggregation." We present data that also supports the hypothesis that adenosine engages a specific cell surface receptor to mediate inhibition of stimulated neutrophils. Theophylline (10 and 100 mu M), a competitive antagonist at adenosine receptors, reversed the effects of adenosine (0.1 mu M) on superoxide anion generation by stimulated neutrophils. The adenosine analogue 5'N-ethylcarboxamidoadenosine (NECA) was a more potent inhibitor of superoxide anion generation than either N6-phenylisopropyladenosine (PIA) or adenosine, an order of potency consistent with that previously demonstrated for adenosine A2 receptors. 2-Chloroadenosine inhibited superoxide anion generation at concentrations similar to NECA. [3H]-NECA and [3H]-2-chloroadenosine bound to a single receptor on intact neutrophils. The characteristics of the receptors for [3H]-NECA and [3H]-2-chloroadenosine were similar (Kd = 0.22 and 0.23 mu M, respectively; number of binding sites = 9.31 and 11.1 X 10(3) sites/cell, respectively). NECA, 2-chloroadenosine, adenosine, and PIA inhibited binding of [3H]-NECA with a rank order similar to that for inhibition of superoxide anion generation (NECA = 2-chloroadenosine greater than adenosine greater than PIA). There was 50% inhibition of superoxide anion generation by NECA at approximately 20% receptor occupancy. Adenosine, derived from damaged tissues, may serve as a specific, endogenous modulator of superoxide anion generation by activated neutrophils through interaction at this newly described receptor on human neutrophils.     

Stimulation of Ca2+-activated human platelet phospholipase A2 by diacylglycerol.

The adenosine analogues 5'-(N-ethyl)carboxamidoadenosine (NECA) and N6-(phenylisopropyl)adenosine (PIA) activate glycogen phosphorylase 5-fold and 4.2-fold respectively in rat hepatocytes incubated in the absence of endogenous adenosine. Half-maximally effective concentrations are 0.5 microM for NECA and 20 microM for PIA, demonstrating the presence of A2-adenosine receptors. Exogenous adenosine activates phosphorylase 4.6-fold, but high rates of adenosine disappearance from the medium render estimates of its half-maximally effective concentration unreliable. These effects of NECA and adenosine are inhibited by 0.1 mM-caffeine. Activation of phosphorylase by a physiological concentration of adenosine (3.3 microM) was 50% inhibited by a physiological concentration of caffeine (35 microM).     

Inhibition by adenosine of reactive oxygen metabolite production by human polymorphonuclear leucocytes.

The stimulation of reactive oxygen metabolite production from human polymorphonuclear leucocytes by chemotactic peptide (fMet-Leu-Phe) was inhibited by adenosine with a K0.5 of 0.6 microM. Dipyridamole (0.1 microM), an inhibitor of adenosine uptake, did not prevent the effect of adenosine. Non-metabolizable analogues could substitute for adenosine in the potency order N-ethoxycarboxamideadenosine greater than 2-chloroadenosine greater than adenosine greater than L-N6-(phenylisopropyl)adenosine = D-N6-(phenylisopropyl)adenosine, which is characteristic of an A2 adenosine receptor. The effects of adenosine, 2-chloroadenosine and N-ethoxycarboxamideadenosine were reversed by 8-phenyltheophylline. When endocytosis was inhibited with cytochalasin B, cells were still susceptible to adenosine receptor agonists. 2-Chloroadenosine (10 microM) reduced the activation of respiration in response to chemotactic peptide from 3.3-fold to 1.4-fold. Activation of reactive oxygen metabolite production in response to latex beads was not reversed by adenosine or its analogues. It was concluded that adenosine acts at an A2 adenosine receptor to antagonize the activation of polymorphonuclear leucocytes by those stimuli, such as chemotactic peptide, which cause an increase in the intracellular free Ca2+ concentration.