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.     

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.     

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.     

The arterial blood supply of the human patella. Its clinical importance for the operating technique in vascularized knee joint transplantations

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK1 and NK2 receptor agonists, [Sar9]substance P (SP) sulfone and [beta Ala8]neurokinin A (NKA) (4-10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 microM) and indomethacin (10 microM). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nM) was selected: [Sar9]SP sulfone (10 nM) produced a biphasic contraction, the two amplitudes averaging 75 +/- 2 and 43 +/- 3% of the maximal response to KCl (80 mM) at 1 and 15 min from application of the agonist, respectively. CPA (3 microM for 60 min) slightly reduced the phasic response to [Sar9]SP sulfone (16 +/- 4% inhibition) and markedly suppressed the tonic component (89 +/- 3% inhibition). 3 The contraction produced by [beta Ala8]NKA (4-10) (10 nM) was more sustained than that induced by the NK1 receptor agonist: it averaged 69 +/- 5 and 73 +/- 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [beta Ala8]NKA (4-10) (18 +/- 7 and 21 +/- 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK1 and NK2 receptor antagonists (SR 140333 and MEN 10627, respectively, 1 microM each) and of L-nitroarginine (100 microM), KCl (40 mM) produced a distinct phasic and tonic contraction which was suppressed by 1 mM nifedipine. CPA (3 microM) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 microM nifedipine, the response to [beta Ala8]NKA (4-10) was slightly depressed (32 +/- 6% inhibition) in its early component only, while the response to [Sar9]SP sulfone was abolished. CPA produced a slight inhibition (15 +/- 9 and 33 +/- 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [beta Ala8]NKA (4-10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [beta Ala8]NKA (4-10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar9]SP sulfone (0.1 microM for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 microM) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar9]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK1 receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK2 receptor-mediated contractile responses.     

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.     

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     

Preliminary validation of the Child Abuse Potential Inventory in Argentina

Experiments were performed to characterize the pharmacology of Sch 50971 ((+)-trans-4-(4(R)-methyl-3(R)-pyrolidinyl)-1H-imidazole dihydrochloride, CAS 167610-28-8), a novel histamine H3 receptor agonist. The activity of Sch 50971 was compared with that of (R)-alpha-methylhistamine (CAS 75614-87-8), a potent and moderately selective agonist of histamine H3 receptors, in a series of in vitro and in vivo assays. Sch 50971 is a high affinity, selective H3 receptor agonist in vitro and in vivo. Sch 50971 inhibits [3H]-N-alpha-methylhistamine (CAS 673-50-7) binding to the histamine H3 receptor in human brain (Ki = 5.0 nmol/l) and guinea pig brain (Ki = 2.5 nmol/l). Sch 50971 also inhibits electric field stimulated guinea pig ileum contractions (pD2 = 7.47) and decreases [3H]-norepinephrine (CAS 51-41-2) release (pD2 = 7.48) from guinea pig pulmonary artery by activation of presynaptic inhibitory H3 receptors. The in vitro effects of Sch 50971 are antagonized by low concentrations of a selective H3 antagonist, thioperamide (CAS 106243-16-7). Sch 50971 has low affinity (IC50's > 10 mumol/l) for histamine H1, dopamine D1 and D2, serotonin 5-HT2 and muscarinic cholinergic receptors. It also does not exhibit histamine H2-antagonist activity. In guinea pigs and cats, Sch 50971 exhibits in vivo H3 agonist activity. Sch 50971 inhibits sympathetic hypertension evoked by stimulation of the medulla oblongata in anesthetized guinea pigs (ED30 = 0.3 mg/kg i.v., ED30 = 1.0 mg/kg i.d.). Sch 50971 also inhibits the effects of sympathetic nerve stimulation on nasal resistance in cats. In these assays, Sch 50971 exhibits an efficacy and potency comparable to H3-agonist (R)-alpha-methylhistamine. However, under in vivo conditions, Sch 50971 does not exhibit histamine H1-mediated responses that are seen with (R)-alpha-methylhistamine at doses close to those that produce H3 effects. Therefore, Sch 50971 is a novel, potent and selective agonist of histamine H3 receptors with an improved in vitro and in vivo receptor profile selectivity compared with (R)-alpha-methylhistamine.     

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 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.     

Efficacy of a program for tobacco use cessation with a combined substitution treatment of nicotine (patches plus chewing gum) at 6 months of follow-up

The compound fedotozine, recently described as a peripheral kappa opioid receptor agonist, was tested on smooth muscle cells isolated from the longitudinal muscle layer of the guinea pig ileum, in comparison with the selective kappa receptor agonist, compound U-50488. Fedotozine (1 nmol/l-1 micromol/l) caused a concentration-dependent contraction of intestinal cells, with a maximum decrease in cell length not significantly different from that caused by acetylcholine. The kappa agonist U-50488 (0.1 pmol/l-100 nmol/l) was without effect. The contractile effect of fedotozine was not significantly modified by naloxone (0.1-1 micromol/l). These results indicate that fedotozine can have direct excitatory effects on intestinal smooth muscle cells from the guinea pig ileum not mediated by activation of kappa opioid receptors.     

Effect of adenosine receptor agonists on spontaneous and K(+)-evoked acetylcholine release from the in vivo rat cerebral cortex

Repeated applications of elevated K+ (100 mM) in artificial cerebrospinal fluid (CSF) were used to evoke an efflux of acetylcholine (ACh) from the in vivo rat cerebral cortex using a cortical cup technique. Elevated K+ reproducibly increased the levels of ACh in cup superfusates by a factor of 3-5-fold above basal levels (27.2 +/- 9.7 nM). The adenosine A1 receptor agonist N6-cyclopentyl adenosine (CPA), at a concentration of 10(-8) M, depressed basal, but not K(+)-evoked ACh efflux. 10(-6) M CPA increased basal, but did not alter K(+)-evoked, ACh efflux. The A2 selective agonist CGS 21680 did not alter either basal, or K(+)-evoked, ACh efflux. The inhibitory effects of 10(-8) M CPA on ACh efflux would be consistent with the presence of adenosine A1 receptors on cholinergic nerve terminals in the cerebral cortex. At a higher concentration (10(-6) M) CPA elevated basal release, possibly by activating low affinity A2 receptors. The failure of CGS 21680 (10(-8) M) to alter basal ACh release suggests an absence of high affinity A2 receptors in these terminals. Whereas elevated K+ in cup superfusates consistently enhanced ACh efflux from the cerebral cortex, this increase was not affected by either CPA or CGS 21680. High K(+)-evoked release of cerebral cortical ACh may be an inappropriate model for the study of adenosine's actions on neurotransmitter release.     

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.     

Adenosine receptor agonists: synthesis and biological evaluation of the diastereoisomers of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA

Among the recently reported 2-(ar)alkynyl derivatives of 5'-N-ethylcarboxamidoadenosine (NECA), the (R,S)-2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA [(R,S)-PHPNECA or SCH 59761] was found to be a very potent agonist at A1 and A2A receptor subtypes, with a Ki of 2.5 nM and 0.9 nM, respectively. Furthermore, this compound showed an inhibitory activity on platelet aggregation 16-fold higher than NECA, being the most potent anti-aggregatory nucleoside reported so far. Since this compound bears a chiral carbon in the side chain, the diastereoisomer separation was undertaken both by chiral HPLC and by a stereospecific synthetic method. Binding assays have shown that the (S)-diastereomer is about fivefold more potent and selective than the (R)-diastereomer as agonist of the A2A receptor subtype [(S)-PHPNECA, KiA2A = 0.5 nM; (R)-PHPNECA, KiA2A = 2.6 nM]. Functional studies indicated that (S)-PHPNECA possesses marked vasodilating activity and produces a relevant decrease in heart rate. Moreover, the (S)-diastereomer proved to be about ten times more potent than the (R)-diastereomer in inducing cardiovascular effects, in in vivo hemodynamic studies. However, the greatest difference between these two enantiomers resulted in the platelet aggregation test: in fact, the (R)-diastereomer displayed an inhibitory activity similar to that of NECA, whereas the (S)-diastereomer was 37-fold more active than NECA as an inhibitor of rabbit platelet aggregation, induced by ADP. These data suggest that (S)-PHPNECA could be a useful tool to investigate the mode of binding of agonists to the platelet adenosine receptor subtype.     

Defibrotide, a single-stranded polydeoxyribonucleotide acting as an adenosine receptor agonist

The binding of single-stranded polydeoxyribonucleotides to adenosine A1 and A2 receptors was investigated. Defibrotide, a natural substance with established anti-thrombotic and anti-ischaemic effects, displaced [3H]CHA (N6-cyclohexyl-adenosine) and [3H]NECA (5'-N-ethylcarboxamido-adenosine) concentration dependently, completely and competitively. Ki values of 371 +/- 68 and 688 +/- 115 micrograms/ml (mean +/- S.E.M. of 4-5 replications) were computed for adenosine A1 and A2 sites, respectively. Higher and lower molecular weight polydeoxyribonucleotides displayed comparable affinity, whereas a double-stranded polydeoxyribonucleotide and a polyanion with a negative charge comparable to that of defibrotide were inactive. Defibrotide did not affect the total number of binding sites in radioligand saturation experiments. Defibrotide relaxed the K(+)-contracted guinea-pig trachealis muscle (IC50 = 4001 micrograms/ml) about one-third as potently as the CHA-contracted preparation and as potently as the resting preparation. NECA, a mixed adenosine A1/A2 receptor agonist, behaved similarly. The effects were abolished by the adenosine A1/A2 receptor blocker 8-phenyltheophylline, but not by the selective A1 blocker, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine. These results demonstrate that defibrotide binds to adenosine receptors and triggers pharmacological responses comparable to those of a known agonist.     

Cross talk between receptors mediating contraction and relaxation in the arterioles but not the dilator muscle of the rat iris

1. Sympathetic nerve stimulation causes contraction of the dilator muscle and the large arterioles of the iris via the activation of alpha 1B-adrenoceptors. We have investigated whether increases in adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and the activation of receptors in these tissues can modulate these nerve-mediated contractions. 2. Increasing intracellular cyclic AMP with dibutyryl cyclic AMP (1 mM), forskolin (50 microM) or isobutylmethylxanthine (100 microM) produced relaxation of both the dilator and the arterioles, abolished the nerve-mediated constriction of the arterioles, but potentiated the nerve-mediated contraction of the iris dilator. 3. Pretreatment of the preparations with cholera toxin, to activate Gs permanently, caused a dilatation of the arterioles and abolished the nerve-mediated constriction but had no effect on the dilator muscle. 4. The beta-adrenoceptor agonist, isoprenaline (1 microM), the adenosine-A1,-A2 agonist, N-ethylcarboxamidoadenosine NECA (100 nM), in the presence of the A1-selective antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 10 nM), and calcitonin gene-related peptide (CGRP, 10 nM) all separately caused a dilatation of the arterioles and abolished the nerve-mediated constriction, while only isoprenaline (1 microM) produced an effect on the dilator, i.e. a relaxation but a potentiation of the nerve-mediated contraction. These results suggest the presence of at least 3 types of receptor linked to Gs and an increase in cyclic AMP in the arterioles, i.e. beta-adrenoceptor, adenosine-A2 and CGRP, but only 1 Gs-linked receptor, i.e. beta-adrenoceptors, on the dilator muscle cells.2+ '     

Nucleotide sequence of the VP7 gene of human rotavirus isolated in Calcutta, India: possible emergence of a new subtype of serotype I

The present study investigated the possible role of nitric oxide (NO) in the development of the withdrawal contractures of guinea pig isolated ileum after acute activation of mu- and kappa-opioid receptors. After a 4-min in vitro exposure to morphine (mu-opioid receptor preferring, but not selective, agonist), [D-Ala2-N-methyl-Phe4-Gly5-ol-]enkephalin (DAMGO; highly selective mu-opioid receptor agonist), or trans(+/-)-3,4-dichloro-N-methyl-N-2(1-pyrrolidynyl)cyclohexyl-ben zeneacetamide (U50-488H; highly selective kappa-opioid receptor agonist), the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. L-N(G)-nitro arginine methyl ester (3-300 microM) injected 10 min before the opioid receptor agonists was able dose dependently to reduce the naloxone-induced contraction after exposure to mu- and kappa-opioid receptor agonists whereas D-N(G)-nitro arginine methyl ester at the same concentrations did not affect it. The inhibitory effect of L-N(G)-nitro arginine methyl ester on morphine, DAMGO and U50-488H withdrawal was dose dependently reversed by L-arginine (3-300 microM) but not by D-arginine. Finally, glyceryl trinitrate on its own (3-300 microM) significantly increased the naloxone-induced contraction after exposure to mu- and kappa-opioid receptor agonist and it was also able to reverse the inhibition of opioid withdrawal caused by L-N(G)-nitro arginine methyl ester. These results provide evidence that NO has a role in the development of opioid withdrawal and that mu- or kappa-opioid receptors are involved.     

Endothelin receptors mediating vasoconstriction in rat pressurized small arteries

The effects of endothelin-1 (ET-1), a nonselective ETA and ETB receptor agonist, and sarafotoxin S6c, a selective ETB agonist, were investigated in the presence and absence of BQ123 and BQ788, ETA- and ETB-selective antagonists, respectively, in rat mesenteric small arteries, using a perfusion pressurized arteriograph in which segments of vessels were cannulated and exposed to constant pressure and flow. ET-1 (10(-13)-10(-7) M) induced vasoconstriction in both intact and endothelium-denuded arteries in a concentration-dependent manner. BQ123 (10(-7) and 10(-6) M) inhibited the effect of ET-1, displacing the concentration-response curve to the right in a concentration-dependent manner. The effect of ET-1 was not significantly affected by BQ788 (10(-7) and 10(-6) M), a selective antagonist of ETB receptors. Sarafotoxin S6c (10(-11)-10(-7) M) also induced a slight concentration-dependent vasoconstriction. The effect of sarafotoxin S6c (10(-8) M) was inhibited by the ETB-selective antagonist BQ788 (10(-7) M), but was not significantly changed by BQ123 (10(-7) M). Vasoconstriction induced by sarafotoxin S6c (10(-8) M) in a single bolus concentration was significantly greater than the contraction induced by the same concentration as part of a cumulative concentration-response curve, indicating desensitization or downregulation of ETB receptors during the latter. Repeated application of single concentrations of sarafotoxin S6c (10(-8) M) caused progressively smaller contraction of arteries. These results show the existence of both ETA and ETB vasoconstrictor receptors located on smooth muscle of small arteries. They also show that ETB receptors induce a smaller constrictor effect, and rapidly undergo desensitization after sustained or repeated activation.     

Potassium-evoked efflux of transmitter amino acids and purines from rat cerebral cortex

Repeated applications of elevated K+ (50 or 75 mM) in cerebral cortical cup superfusates was used to evoke an efflux of gamma-aminobutyric acid (GABA), glutamate, aspartate, glycine, adenosine, and inosine from the in vivo rat cerebral cortex. K+ (50 mM) significantly elevated GABA levels in cup superfusates but had little effect on the efflux of glutamate, aspartate, glycine, adenosine, or inosine. K+ (75 mM) significantly enhanced the efflux of GABA, aspartate, adenosine, and inosine and caused nonsignificant increases in glutamate and glycine efflux. The adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA), applied in cup superfusates at a concentration of 10(-10) M had no effect on either basal or K(+)-evoked release of any of the amino acids or purines measured. At 10(-6) M CPA significantly enhanced aspartate release, and depressed GABA efflux. The selective A2 adenosine receptor agonist 2-p(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS 21680) (10(-8) M) was without effect on either basal, or K(+)-evoked, efflux of amino acids or purines. The enhancement of aspartate (an excitotoxic amino acid) efflux by higher concentrations of CPA is likely due to activation of adenosine A2b receptors. This observation may be of relevance when selecting adenosinergic agents to treat ischemic or traumatic brain injuries. Overall, the results suggest that effects of adenosine receptor agonists on K(+)-evoked efflux of transmitter amino acids from the in vivo rat cerebral cortex may not be comparable to those observed with in vitro preparations.     

Tachykinins and reflexly evoked atropine-resistant motility in the guinea pig colon in vivo

Distension of a balloon placed in the proximal colon of anesthetized, guanethidine- and naloxone-pretreated guinea pigs elicited a series of long-lasting regular phasic pressure waves which were suppressed by hexamethonium. Activity evoked by a low degree of balloon distension was largely, but not completely, suppressed by atropine. Further balloon distension in atropine-treated animals enabled us to study the effect of tachykinin receptor antagonists on the atropine-resistant and hexamethonium-sensitive response to distension. The selective tachykinin receptor antagonists, (+/-)-CP 96,345 for the NK-1 receptor and L 659,877, MEN 10,376 and SR 48,968 for the NK-2 receptor, inhibited with varying potency the atropine-resistant response to distension. These antagonists also blocked the contraction of the guinea pig colon produced by the i.v. administration of selective NK-1 and NK-2 receptor agonists. In vitro experiments, using mucosa-free circular muscle strips from the guinea pig colon, proved the existence of functional NK-1 and NK-2 receptors in this tissue. We conclude that both NK-1 and NK-2 receptors participate in the atropine-resistant reflex contractions produced by localized balloon distension of the guinea pig colon in vivo.     

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)