Effects of exogenous phytohormones on plastid tRNA modifications in ragi coleoptiles

The influence of adenosine and selective A1 and A2 agonists and antagonists was investigated on the cholinergic and the excitatory non-cholinergic (e-NC) contractions induced by electrical field stimulation in the guinea-pig bronchi. Adenosine (10 nM-1 mM) induced a concentration-dependent inhibition of the e-NC contraction (EC50 = 90 +/- 14 microM), whereas the cholinergic peak was only slightly affected. Preincubation of the tissue with the adenosine uptake blocker dipyridamole (10 microM) significantly shifted the concentration-inhibition curve to adenosine to the left (EC50 = 10 +/- 1 microM), suggesting an interaction with extracellular adenosine receptors of A1 and/or A2 subtype. To characterize the receptor type involved in this effect, selective adenosine derivatives were studied. The agonist to both A1 and A2 adenosine receptors, 5'-N-ethylcarboxamidoadenosine (NECA) was more potent than the selective A1 agonist, (-)-R-6-phenylisopropyladenosine (R-PIA), in inhibiting the e-NC contraction (EC50 = 0.10 +/- 0.04 and 0.60 +/- 0.12 microM, respectively, with a maximal inhibition of 70 and 45%, respectively). The concentration-response curve to NECA was shifted to the right by the A2 receptor selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (10 microM) (EC50 = 1.4 +/- 0.5 microM) as well as by the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 microM) (EC50 = 0.7 +/- 0.3 microM). The inhibitory effect induced by the association of both antagonists, DPCPX and DMPX, was considerably potentiated (EC50 > 22 +/- 2.5 microM). The effect of R-PIA was also shifted to the right by DPCPX (EC50 = 8.2 +/- 1.6 microM) but was not modified by DMPX. The contractile response to exogenous substance P was unaffected by NECA pretreatment (0.3 microM). Altogether, these results suggest that adenosine-induced inhibition of e-NC contraction of guinea-pig bronchi is mediated through activation of both A1 and A2 adenosine receptors linked to inhibition of the release of neuropeptides from C-fibre nerve endings.     

Characterization of ocular hypertension induced by adenosine agonists

PURPOSE: Previous studies have shown that adenosine agonists may induce a rise in intraocular pressure (IOP), a reduction in IOP, or both. Although the reduction in IOP results from the activation of adenosine A1 receptors, the mechanisms responsible for the rise in IOP have not been investigated. This study examines the receptors and mechanisms responsible for the adenosine agonist-induced rise in IOP. METHODS: The ocular effects of the nonselective adenosine agonist NECA, the relatively selective adenosine A2 agonist CV-1808, the A2a agonist CGS-21680, and the A1 agonist R-PIA were evaluated. RESULTS: The topical administration of CV-1808 produced a rapid rise in IOP, with a maximum increase of 15.6 +/- 1.6 mm Hg. Dose-response curves demonstrated that each agonist produced a dose-related rise in IOP with the following rank order of potency: NECA > CV-1808 > > R-PIA = CGS-21680. At times corresponding to the rise in IOP, the administration of high doses of CV-1808 (165 micrograms) produced a significant increase in aqueous humor flow and protein concentration. Increases in IOP and aqueous humor protein levels induced by CV-1808 were blocked by pretreatment with the adenosine A2 antagonist DMPX. In vitro studies demonstrated that CV-1808 did not alter cyclic adenosine monophosphate production in the rabbit iris-ciliary body. In cats, topical administration of CV-1808 produced a rapid rise in IOP, with a maximum increase of 8.1 +/- 2.4 mm Hg and an ED50 of 73 +/- 2.9 micrograms. This rise in IOP was blocked by DMPX pretreatment. CONCLUSIONS: These data demonstrate that adenosine receptor agonists can induce an acute rise in IOP in rabbits and cats. On the basis of pharmacologic characteristics, the rise in IOP is consistent with the activation of ocular adenosine A2 receptors. Functional studies indicate that at high doses, this rise in IOP involves an increase in aqueous flow and the breakdown of the blood-aqueous barrier.     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM). 2. In the prostatic half of the vas deferens the A1 selective adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A1/A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC50+/-s.e.mean values 6.15+/-0.24, 5.99+/-0.26 and 5.51+/-0.24, respectively). The responses to CPA were blocked by the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM). 3. In the epididymal half of the vas deferens NECA potentiated (at < or = 100 nM) and inhibited (at > or = 1 microM) electrically-evoked contractions. In the presence of the non-selective alpha-adrenoceptor antagonist phentolamine (3 microM), the alpha1-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC50 values 6.05+/-0.25, 5.97+/-0.29 and 5.71 +/-0.27, respectively). CPA (at 10 microM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC50 6.14+/-0.67); this effect was antagonized by DPCPX (30 nM, apparent pK(B) 8.26+/-0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 microM), CPA (up to 1 microM) potentiated electrically-evoked contractions. 4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC50 values 7.49+/-0.62, 7.65+/-0.74 and 5.84+/-0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK(B) value of 7.64+/-0.64. 5. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 microM) potentiated responses to phenylephrine (< or = 1 microM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 microM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 microM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC50 values 7.57+/-0.54 and 8.08+/-0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis. 6. These studies are consistent with the action of stable adenosine analogues at prejunctional A1 and postjunctional A1-like adenosine receptors. The prejunctional A1 adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of alpha1-adrenoceptor-, but not of ATP-induced contractility.     

Adenosine analogues with specificity for A2 receptors bind to mouse spermatozoa and stimulate adenylate cyclase activity in uncapacitated suspensions

Adenosine and its analogues, known to stimulate adenylate cyclase activity in somatic cells via A2 receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. Indirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed functional responses. In the present study we provide evidence that [3H]5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with specificity for stimulatory A2 adenosine receptors, can bind to mouse spermatozoa. This binding can be displaced by both unlabelled NECA and 2-chloroadenosine, another A2 receptor agonist, but not by cyclopentyladenosine, an inhibitory A1 receptor agonist, suggesting that the NECA binding is specific for A2 receptors. The presence of S-(p-nitrobenzyl)-6-thioinosine, an adenosine transport inhibitor, did not affect binding, indicating an external site for interaction with sperm cells. Saturable specific binding of [3H]NECA to mouse spermatozoa incubated at 37 degrees C was observed, with a Bmax of 5.17 pmol mg-1 protein and a Kd value of 930 nmol l-1. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstrable binding of [3H]NECA, both NECA and 2-chloroadenosine significantly stimulated adenylate cyclase activity in a concentration-dependent manner, with NECA being effective at a lower concentration. Furthermore, the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the presence of either NECA or 2-chloroadenosine, also significantly stimulated enzyme activity. In somatic cells, expression of responses to adenosine usually requires GTP and G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of multiple sites by adenosine analogues in the rat isolated aorta

1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100 microM) E/[A] curves to NECA and CPA were right-shifted. However, no further shift of the CPA E/[A] curve was obtained when 8-SPT (50 microM) was administered concomitantly. The locations of curves to R-PIA and adenosine were unaffected by L-NAME (100 microM). 6. In the presence of PD 115,199 (0.1 microM) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 +/- 0.19). PD 115,199 (0.1 and 1 microM) gave smaller rightward shifts of E/[A] curves to R-PIA and CPA, but E/[A] curves to adenosine were not significantly shifted in the presence of PD 115,199 (0.1 or 1 microM). 7. The presence of ZM 241385 (3 nM-0.3 microM) caused parallel rightwad shifts of NECA E/[A] curves (pKB = 8.73 +/- 0.11). No significant shifts of E/[A] curves to adenosine, CPA or R-PIA were observed in the presence of 0.1 microM ZM 241385. 8. CGS 21680 (1 microM) elicited a relaxant response equivalent to approximately 40% of the NECA maximum response. In the presence of this concentration of CGS 21680, E/[A] curves to NECA were right-shifted in excess of 2-log units, whereas E/[A] curves to R-PIA were not significantly shifted. 9. BWA1433 (100 microM) caused a small but significant right-shift of the E/[A] curve to R-PIA yielding a pA2 estimate of 4.1 IB-MECA (N6-(3-iodo-benzyl)adenosine-5(1)-N-methyl uronamide) elicited relaxant responses which were resistant to blockade by 8-SPT (p[A]50 = 5.26 +/- 0.13). 10. The results suggest that whereas relaxations to NECA (10 nM-1 microM) are mediated via adenosine A2a receptors, which are located at least in part on the endothelium, R-PIA and CPA may activate A2b receptors on the endothelium and an additional, as yet undefined site, which is likely to be located on the smooth muscle and which is not susceptible to blockade by 8-SPT, PD 115,199 or ZM 241385. This site is unlikely to be an A3 receptor since the very small shift obtained in the presence of BWA1433 (100 microM), and the low potency of IB-MECA is not consistent with the affin     

Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools

Presynaptic adenosine receptors inhibit transmitter release at many synapses and are known to exist on retinotectal terminals. In this paper we show that adenosine decreases retinotectal field potentials by approximately 30% and investigate the mechanism. First, as judged by the effects of specific calcium channel blockers, retinotectal transmission was mediated almost exclusively by N-type calcium channels, which are known to be modulated by adenosine A1 receptors. Transmission was completely blocked by either omega-Conotoxin GVIA (-100%, N-type blocker) or omega-Conotoxin MVIIC (-99%, N-, P- and Q-type blocker) and was not significantly affected by omega-Agatoxin IVA [+1.7 +/- 9. 3% (SE), P-,Q-type blocker], but was augmented slightly by nifedipine(+9.3 +/- 2.1%, L-type blocker). Second, the adenosine inhibition was presynaptic, as indicated by a 43% increase in paired-pulse facilitation. Third, the selective A1 agonist cyclohexyl adenosine (CHA) at 50 nM caused a 21% decrease in amplitude and the selective A2 agonist N6-[2-(3, 5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) at 100 nM caused a 24% increase. Fourth, the selective A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) alone produced an increase in the field potential, suggesting a tonic inhibition mediated by endogenous adenosine. Fifth, pertussis toxin eliminated adenosine inhibition implicating Gi or Go protein coupling. Sixth, C-kinase activation eliminated the A1-mediated inhibition. In regenerating projections, adenosine also caused a decrease in transmission (-30 +/- 12%), but after induction of long-term potentiation (LTP) via trains of stimuli or via treatment with the phosphatase inhibitor okadaic acid, the adenosine response was converted to an augmentation. Because LTP is associated with C-kinase activation, this is consistent with C-kinase uncoupling the A1 receptor from inhibiting N-type Ca2+ channels. This uncovers the A2-mediated augmentation as demonstrated in normals with DPMA. Such an effect could account in part for the LTP of immature synapses and the change from rapidly fatiguing to robust synaptic transmission.     

Genetic risk of neuropsychological impairment in schizophrenia: a study of monozygotic twins discordant and concordant for the disorder

We investigated the effect of the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30-40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 +/- 3.3%), slowly developing inhibition of [Ca2+]i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca2+]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca2+ influx pathway (e.g., L-type Ca2+ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

Photoaging and tretinoin

BACKGROUND: Adenosine is a potent vasodilator of vascular smooth muscle. Endothelium-derived nitric oxide (NO) elicits vasodilation. We have previously reported that adenosine stimulates the production of NO from porcine carotid arterial endothelial cells (PCAEC) via a receptor-mediated mechanism. This study was to determine whether adenosine also enhances NO production from human arterial endothelium and to define the involvement of adenosine A1 and A2 receptors. MATERIALS AND METHODS: Human iliac arterial endothelial cells (HIAEC) and PCAEC were harvested and cultured in dishes. NO production was evaluated with a NO electrode sensor which measured continuously real-time NO production. RESULTS: NO content of the medium bathing HIAEC and PCAEC was significantly increased with adenosine (100 micromol/L). Ethylcarboxamidoadenosine (NECA), a nonselective adenosine receptor agonist, and carboxyethyl-phenethylamino-ethylcarboxamidoadenosine (CGS-21680), a selective adenosine A2a receptor agonist, increased NO production by HIAEC and PCAEC with respective EC50 values of 3.32 and 6.96 nmol/L for NECA and 30.97 and 29.47 nmol/L for CGS-21680. Chlorofuryl-triazolo-quinazolinamine (CGS-15943; 1 micromol/L), an adenosine A1 and A2 receptor antagonist, and aminofuryltriazolotriazinyl-aminoethylphenol (ZM-241385; 1 micromol/L), a selective adenosine A2a receptor antagonist, inhibited the effect of CGS-21680. Chlorocyclopentyl-adenosine (CCPA; 1 micromol/L), an adenosine A1 receptor agonist, significantly depressed NO production by both HIAEC and PCAEC: This effect was inhibited by cyclopentyl-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist. CONCLUSIONS: The results demonstrate that adenosine A2a receptors increase, and adenosine A1 receptors decrease, the production of NO by human and porcine arterial endothelial cells.     

The endothelium of the rat renal artery plays an obligatory role in A2 adenosine receptor-mediated relaxation induced by 5'-N-ethylcarboxamidoadenosine and N6-cyclopentyladenosine

Studies were undertaken in the rat isolated renal artery in order to determine if adenosine receptor agonists were capable of inducing the release of nitric oxide from the renovascular endothelium. N6-cyclopentyladenosine (CPA) and 5'-N-ethylcarboxamidoadenosine (NECA) produced concentration-dependent relaxations in endothelium intact renal artery rings. The NECA curve was biphasic with a first phase pA50 of 6.05. The CPA curve was monophasic with a pA50 of 4.35. In the absence of endothelium the curves to both NECA and CPA were monophasic with pA50 values of 3.37 and 3.50, respectively. The A2a adenosine receptor-selective agonist CGS21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenos ine) was inactive in endothelium intact tissues. Relaxant responses to CPA and NECA in the presence of endothelium were antagonized by 8-p-sulfophenyltheophylline and by 1,3-dipropyl-8-cyclopentylxanthine only at a nonselective concentration (3 x 10(-6) M) suggesting activation of A2 adenosine receptors. The responses to CPA and NECA in the absence of endothelium are not due to activation of A1 or A2 adenosine receptor subtypes because they are resistant to blockade by these xanthines. CPA and NECA responses in the presence of endothelium were inhibited by NG-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase inhibitor, but not by the cyclooxygenase inhibitor indomethacin or the K+ATP channel antagonist glibenclamide. These results suggest that the rat renal artery contains A2b adenosine receptors that are located exclusively on the endothelium and cause the release of nitric oxide.     

The foot in hereditary motor and sensory neuropathies in children

We investigated the regulation of COX-2 expression and activity by adenosine receptors in rat microglial cells. The selective adenosine A2a-receptor agonist CGS21680 and the non-selective adenosine A1- and A2-receptor agonist 5'-N-ethylcarboxiamidoadenosine (NECA) induced an increase in COX-2 mRNA levels and the synthesis of prostaglandin E2 (PGE2). The adenosine A1-receptor agonist cyclopentyladenosine (CPA) was less potent, and the adenosine A1-receptor-specific agonist N6-2-(-aminophenylo)ethyladenosine (APNEA) showed only marginal effects. Microglia expressed adenosine A1-, A2a-, and A3-, but not A2b-receptor mRNAs, whereas astroglial cells expressed adenosine A2b- but not A2a-receptor mRNA. The adenosine A2a-receptor selective antagonist (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) inhibited both CGS21680-induced COX-2 expression and PGE2 release. CGS21680-increased PGE2 levels were inhibited by dexamethasone, by the nonsteroidal antiinflammatory drug meloxicam, and by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CGS21680 and NECA both increased intracellular cAMP levels in microglial cells. Dibutyryl cAMP as well as forskolin induced the release of PGE2. The results strongly suggest that adenosine A2a-receptor-induced intracellular signaling events cause an up-regulation of the COX-2 gene and the release of PGE2. Apparently, the cAMP second messenger system plays a crucial role in COX-2 gene regulation in rat microglial cells. The results are discussed with respect to neurodegenerative disorders of the CNS such as Alzheimer's disease, in which activated microglia are critically involved and COX inhibitors may be of therapeutic benefit.     

Adenosine receptor-mediated relaxation of guinea-pig precontracted, isolated trachea

1. We have investigated the pharmacological profile of the adenosine receptor mediating relaxation of the carbachol pre-contracted guinea-pig trachea. 2. 5'-N-Ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine elicited concentration-dependent relaxations with pD2 (-log10 half-maximal values) of 6.37 +/- 0.04 and 5.25 +/- 0.09, with maximal relaxations of 73 +/- 7 and 208 +/- 38%, respectively. In the presence of 10 microM NECA, 2-chloroadenosine was able to relax the tissue further with a pD2 value of 4.74 +/- 0.11 and a maximal response of 252 +/- 68%. 3. CGS 21680, APEC and adenosine failed to elicit significant relaxations of precontracted tracheal rings at concentrations below 10 microM. At 10 microM, adenosine analogues elicited relaxations with the following order of magnitude (% relaxation): 2-chloroadenosine (75 +/- 16%) = NECA (69 +/- 16%) > APEC (25 +/- 8%) > CGS 21680 (11 +/- 2%) > adenosine (6 +/- 4%). 4. NECA-induced relaxation of precontracted trachea was antagonized by adenosine receptor antagonists with the rank order of apparent affinity (Ki, nM): PD 115,199 (27 +/- 8) = XAC (43 +/- 11) > CP 66,713(285 +/- 89) = DPCPX (316 +/- 114). 5. We conclude that the adenosine analogue-induced relaxation of guinea-pig tracheal rings fails to fit into the current classification of A2 adenosine receptors.     

Adenosine A2A receptors facilitate 45Ca2+ uptake through class A calcium channels in rat hippocampal CA3 but not CA1 synaptosomes

In the hippocampus, the neuromodulatory role of adenosine depends on a balance between inhibitory A1 responses and facilitatory A2A responses. Since the presynaptic effects of hippocampal inhibitory A1 adenosine receptors are mostly mediated by inhibition of Ca2+ channels, we now investigated whether presynaptic facilitatory A2A adenosine receptors would modulate calcium influx in the hippocampus. The mixed A1/A2 agonist, 2-chloroadenosine (CADO; 1 microM) inhibited veratridine (20 microM)-evoked 45Ca2+ influx into hippocampal synaptosomes of the CA1 or CA3 areas by 24.2 +/- 4.5% and 17.2 +/- 5.8%, respectively. In the presence of the A, antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), the inhibitory effect of CADO (1 microM) on 45Ca2+ influx was prevented in CA1 synaptosomes, but was converted into a facilitatory effect (14.2 +/- 6.7%) in CA3 synaptosomes. The A2A agonist, CGS 21680 (3-30 nM) facilitated 45Ca2+ influx in CA3 synaptosomes, with a maximum increase of 22.9 +/- 3.9% at 10 nM, and was virtually devoid of effect in CA1 synaptosomes. This facilitatory effect of CGS 21680 (10 nM) in CA3 synaptosomes was prevented by the A2A antagonist 8-(3-chlorostyryl)caffeine (CSC; 200 nM), but not by the A1 antagonist, DPCPX (20 or 100 nM). The facilitatory effect of CGS 21680 on 45Ca2+ uptake by CA3 synaptosomes was prevented by the class A calcium channel blocker, omega-agatoxin-IVA (200 nM). These results indicate that presynaptic adenosine A2A receptors facilitate calcium influx in the CA3 but not the CA1 area of the rat hippocampus through activation of class A calcium channels.     

Modulation of erythropoietin production by selective adenosine agonists and antagonists in normal and anemic rats

Hypoxia or anemia is the fundamental stimulus for erythropoietin (EPO) production. Recent in vitro studies suggest that EPO secretion in response to hypoxia is regulated by adenosine in the kidney. In order to examine the in vivo effect of adenosine on EPO production, we determined the effects of adenosine receptor agonists and antagonists on serum EPO concentration in normal and anemic rats. In normal rats, intravenous injection of adenosine agonists (NECA, CHA and CGS-21680) dose-dependently stimulated EPO production. Pretreatment with KW-3902, an adenosine A1 antagonist with modest A2b antagonistic action, or KF17837, an adenosine A2a antagonist, inhibited the NECA (0.1 mg/kg, i.v.)-stimulated EPO production. Anemic hypoxia, induced by 2% (v/w body weight) blood withdrawal, increased serum EPO concentration from 38 +/- 2 to 352 +/- 76 mU/ml, with the increased serum adenosine concentration in the renal vein. KF17837 (0.1 mg/kg, i.v.), but not KW-3902 (0.1 mg/kg, i.v.), inhibited the anemic hypoxia-induced increase in EPO production. The present findings support the notion that adenosine mediates the EPO production in response to hypoxia in the kidney.     

Characterization of adenosine receptors evoking excitation of mesenteric afferents in the rat

We examined the effects of adenosine receptor agonists and antagonists on the discharge of mesenteric afferent nerves supplying the jejunum in pentobarbitone sodium-anaesthetized rats. Adenosine (0.03-10 mg kg(-1), i.v.), NECA (0.3-300 microg kg(-1), i.v.) and the A1 receptor agonist, GR79236 (0.3-1000 microg kg(-1), i.v.), each induced dose-dependent increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. The A1 receptor antagonist, DPCPX (3 mg kg(-1), i.v.), antagonized all the effects of GR79236 but only the haemodynamic effects of adenosine and NECA. The A2A receptor antagonist, ZM241385 (3 mg kg(-1), i.v.), antagonized the hypotensive effect of NECA but none of the effects of GR79236. The A2A receptor agonist, CGS21680 (0.3-300 microg kg(-1), i.v.), and the A3 receptor agonist, IB-MECA (0.3-300 microg kg(-1), i.v.), each induced only a dose-dependent hypotension. Subsequent administration of adenosine (3 mg kg(-1), i.v.) induced increases in afferent nerve activity and intrajejunal pressure and bradycardia. ZM241385 (3 mg kg(-1), i.v.) antagonized the hypotensive effect of CGS21680 but not the effects of adenosine. Bethanechol (300 microg kg(-1), i.v.) evoked increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. However, adenosine (3 mg kg(-1), i.v.) evoked greater increases in afferent nerve activity than bethanechol despite inducing smaller increases in intrajejunal pressure. In summary, A1 and A2B and/or A2B-like receptors evoke adenosine-induced increases in mesenteric afferent nerve activity and intrajejunal pressure in the anaesthetized rat. Furthermore, elevations in intrajejunal pressure do not wholly account for adenosine-evoked excitation of mesenteric afferent nerves.     

Effect of adenosine receptor agonists on release of the nucleoside analogue [3H]formycin B from cultured smooth muscle DDT1 MF-2 cells

Adenosine has receptor-mediated effects in a variety of cell types and is predominantly formed from ATP by a series of nucleotidase reactions. Adenosine formed intracellularly can be released by bidirectional nucleoside transport processes to activate cell surface receptors. We examined whether stimulation of adenosine receptors has a regulatory effect on transporter-mediated nucleoside release. DDT1 MF-2 smooth muscle cells, which possess nitrobenzylthioinosine-sensitive (ES) transporters as well as both adenosine A1 and A2 receptors, were loaded with the metabolically stable nucleoside analogue [3H]formycin B. N6-cyclohexyladenosine (CHA), a selective adenosine A1 receptor agonist, produced a concentration-dependent inhibition of [3H]formycin B release with an IC50 value of 2.7 microM. Further investigation revealed CHA interacts directly with nucleoside transporters with a Ki value of 3.3 microM. Neither 5'-N-ethylcarboxamidoadenosine (NECA), a mixed adenosine A1 and A2 receptor agonist, nor CGS 21680, a selective adenosine A2A receptor agonist, affected nucleoside release. We conclude that release of the nucleoside formycin B from DDT1 MF-2 cells is not regulated by adenosine A1 or A2 receptor activation.     

Evidence for the presence of both P1 and P2 purinoceptors in the rat myometrium

1. Adenosine, adenosine triphosphate (ATP) and some stable analogues of adenosine inhibited field stimulation-induced contractions of the uterus from rats treated with oestradiol cypionate (20 micrograms/kg, s.c.) 1 day previously. Adenosine was twice as potent as ATP; both were potentiated by dipyridamole (10 mumol/L). 2. The order of agonist potency of adenosine and its analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) > N6-cyclohexyladenosine > or = R-phenylisopropyladenosine = S-phenylisopropyladenosine = 2-chloroadenosine > or = adenosine > or = ATP > > 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine. This order suggests the presence of P1 purinoceptors of the A2B subtype. 3. Responses to agonists were antagonized to differing extents by the P1 purinoceptor antagonist 8-phenyltheophylline (10 mumol/L). 4. In uterine preparations from rats pretreated for 2 days with oestrogen (20 micrograms/kg, s.c.) and for 1 day with progesterone (3 mg/animal, s.c.), the inhibitory potencies of adenosine and NECA were reduced, indicating hormonal regulation of uterine responsiveness to P1 purinoceptor agonists. 5. Stable analogues of ATP caused contractions of unstimulated myometrial preparations from oestrogen-treated animals, indicating activation of a P2 purinoceptor, possibly of the P2X subtype, because of the relative order of potency was alpha, beta-methylene ATP > beta, gamma-methylene ATP = ATP = 2-methylthio ATP.     

Functional characterization of adenosine receptors in the nucleus tractus solitarius mediating hypotensive responses in the rat

1. The aim of this study was to characterize adenosine receptors located in the nucleus tractus solitarius (NTS) that mediate decreases in blood pressure in the anaesthetized rat. To determine the adenosine receptor subtype involved, a range of selective agonists and antagonists were studied and their relative potencies evaluated. 2. The rank order of agonist potency in inducing decreases in diastolic blood pressure was N6-cyclopentyladenosine (CPA) > N6-cyclohexyladenosine (CHA) > N-ethyl-carboxamidoadenosine (NECA) > or = 2-phenylaminoadenosine (CV1808) > 2-p-(carboxyethyl)phenethylamino-5' N-ethylcarboxamidoadenosine (CGS 21680) > N6-(2-(4-aminophenyl)ethyl)-adenosine (APNEA). 3. The hypotensive action of CPA following microinjection into the NTS was antagonized by i.v. infusions (50 micrograms kg-1 min-1) of adenosine receptor antagonists, 8-cyclopentyl-1,3 dipropylxanthine (DPCPX), 8-phenyltheophylline (8-PT), 8-(p-sulphophenyl)theophylline (8-SPT), and 1,3-dipropyl-8-N-(2-diethylamino)ethyl)-N methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo) benzenesulphonamidexanthine (PD 115199). The antagonist potency order was DPCPX > PD115199 > or = 8-PT. Intravenous infusion of 8-SPT had no effect on blood pressure responses to microinjection of CPA into the NTS. 4. The results suggest that adenosine A1 receptors in the NTS mediate hypotensive responses in the anaesthetized rat preparation.     

Further investigations into adenosine A1 receptor-mediated contraction in rat colonic muscularis mucosae and its augmentation by certain alkylxanthine antagonists

1. The alkylxanthine antagonists, 8-phenyltheophylline (8-PT), 8-p-sulphophenyltheophylline (8-SPT) and 1,3,7-trimethylxanthine (caffeine) produced rightward displacements of contractile concentration-effect curves to 5'-N-ethylcarboxamidoadenosine (NECA) in rat isolated colonic muscularis mucosae (RCMM) with concentration-ratios consistent with adenosine receptor blockade. The non-xanthine antagonist, 9 fluro-2-(2-furyl)-5,6-dihydro [1,2,4] triazo to [1,5-c]-quinazin-imine (CGS15943A) also antagonized contractions to NECA with an affinity (pKB8.1-8.5) consistent with adenosine A1 receptor blockade. 2. In addition to producing rightward shifts of the concentration-response curves, the maximum contractions to 5'-N-ethylcarboxamidoadenosine (NECA) were also markedly increased in the presence of 8-PT (by 83 +/- 16% at 1 microM), 8-SPT (by 37 +/- 7% at 10 microM) and caffeine (by 45 +/- 5% at 100 microM) but were unaffected by CGS15943A (at 0.01 and 0.03 microM). 3. As with NECA, the maximum contractions to the adenosine A1 receptor agonists R-phenylisopropyladenosine (R-PIA) and N-[(1S, trans)-2-hydroxyclopentyl] adenosine (GR79236) were both antagonized and augmented by 8-PT. In addition, the contractions to NECA in the presence of 8-PT (1 microM) were inhibited by nanomolar concentrations of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 4. The non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (1 microM) produced a marked increase in the NECA maximum without producing a rightward shift in the NECA curve, whereas a higher concentration (10 microM) virtually abolished responses. The PDE type III inhibitor,milrinone (1 microM), the type IV inhibitor, rolipram (10 microM), and the type V PDE inhibitor, zaprinast(3 microM), were all without effect on NECA responses in RCMM.5. Partial inhibitions of contractions to NECA were produced by indomethacin (at 3 or 10 micro M) or piroxicam (at 3 microM). Responses to GR79236 were also partially inhibited by indomethacin. In the presence of indomethacin, 8-PT was still able to enhance markedly the maximum contractions obtained to NECA in RCMM.6. The present study has shown that certain alkylxanthine antagonists (but not the non-xanthineCGS15943A) produced a marked augmentation of adenosine Al receptor-mediated contractions inRCMM. The mechanism of this augmentation is, as yet, not known but is unlikely to result from inhibition of PDE. This study has also shown that adenosine Al receptor-induced contractions inRCMM are mediated, in part, via products of the cyclo-oxygenase pathway.     

Adenosine receptor mediated stimulation of intracellular calcium in acutely isolated astrocytes

The characteristics of adenosine receptors found in glial fibrillary acid protein (GFAP)-positive astrocytes acutely isolated from the cerebral cortices of 4- to 12-day old rats were examined by evaluating the effects of adenosine and its analogues on intracellular calcium levels. First, these effects were compared with those seen in primary astrocytic cultures, and it was found that acutely isolated astrocytes showed much greater sensitivity to adenosine than their cultured counterparts. Then, the adenosine evoked calcium responses in acutely isolated cells were evaluated under various conditions. The responses to adenosine were not inhibited by papaverine, an uptake blocker, or by removal of extracellular calcium. U73122, a phospholipase C inhibitor, was able to completely inhibit the adenosine response. The receptor inhibitor 3-isobutyl-1-methylxanthine inhibited the calcium response to adenosine, providing evidence that the response is not coupled to the xanthine-insensitive A3 receptor. The stimulatory action of NECA, a non-selective analogue, was blocked neither by the A2A-selective receptor antagonist 8-(3-chlorostyryl) caffeine nor by the A1-selective receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The A2B receptor antagonist alloxazine, however, was able to completely inhibit the increase in intracellular calcium produced by NECA. Taken together, these data suggest that the adenosine-evoked calcium response in acutely isolated astrocytes is coupled to the A2B receptor.     

8-(3-Chlorostyryl)caffeine (CSC) is a selective A2-adenosine antagonist in vitro and in vivo

An adenosine antagonist, 8-(3-chlorostyryl)caffeine (CSC), was shown previously to be 520-fold selective for A2a-adenosine receptors in radioligand binding assays in the rat brain. In reversing agonist effects on adenylate cyclase, CSC was 22-fold selective for A2a receptors in rat phenochromocytoma cells (Kb 60 nM) vs. A1 receptors in rat adipocytes (Kb 1.3 microM). Administered i.p. in NIH mice at a dose of 1 mg/kg, CSC shifted the curve for locomotor depression elicited by the A2a-selective agonist APEC to the right (ED50 value for APEC shifted from 20 micrograms/kg i.p. to 190 micrograms/kg). CSC had no effect on locomotor depression elicited by an ED50 dose of the A1-selective agonist CHA. CSC alone at a dose of 5 mg/kg stimulated locomotor activity by 22% over control values. Coadministration of CSC and the A1-selective antagonist CPX, both at non-stimulatory doses, increased activity by 37% (P < 0.001) over CSC alone, suggesting a behavioral synergism of A1- and A2-antagonist effects in the CNS.