Effects of arecoline and cholinesterase inhibitors on cyclic guanosine 3′,5′-monophosphate and adenosine 3′,5′-monophosphate in mouse brain

Summary In mice, Arecoline in vivo dose-dependently increased the cGMP concentrations of the cerebellum and the cerebrum (= parts of cortex, hippocampus, hypothalamus, thalamus, striatum and midbrain) without influencing the cAMP levels. The cholinesterase inhibitors paraoxon and physostigmine caused an elevation only in cerebrum, whereas the cGMP content of the cerebellum even decreased.Pretreatment with atropine prevented the rise in cGMP levels as well as the symptoms of cholinergic stimulation elicited by arecoline or paraoxon. Diazepam reduced cGMP levels below control values and blocked the effect of arecoline, while typical symptoms due to arecoline, e.g., tremor and salivation remained unaffected. The tripeptide prolyl-leucyl-glycinamide (MIF) had no effect on either cGMP values or the peripheral signs of cholinergic stimulation elicited by arecoline.The results show that elevation of cGMP in the central nervous system caused by cholinomimetic agents can be prevented not only by cholinolytics, blocking muscarinic receptors but also by influencing other mechanisms to be discussed.     

Functional roles of cyclic guanosine 3′,5′-monophosphate analogue in cerebral vasodilation

• 1.1. Vasodilating effects of cyclic nucleotides in cerebral vasculature were examined using membrane permeable cyclic nucleotide analogues, 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) and 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP).
• 2.2. In isolated canine basilar artery (CBA), 8-Br-cGMP but not 8-Br-cAMP, significantly inhibited Ca²⁺-induced and agonist [serotonin(5-HT), prostaglandin(PG)F_2α or endothelin]-induced contraction, in a concentration-dependent manner.
• 3.3. When Ca²⁺ was depleted from intracellular store sites by pretreatment with A23187, 8-Br-cGMP but not 8-Br-cAMP strongly attenuated contractions induced by Ca²⁺-influx.
• 4.4. Neither 8-Br-cGMP nor 8-Br-cAMP modified contraction induced by caffeine which elicits Ca²⁺ release from intracellular Ca²⁺ store.
• 5.5. 8-Br-cGMP lowered the high K⁺-induced sustained [Ca²⁺] elevation.
• 6.6. These results suggest that, at least in CBA, cGMP exerts its inhibitory effect on the contraction induced by influx of Ca²⁺, by reducing the level of [Ca²⁺]_i and reducing [Ca²⁺]_i sensitivity of the contractile machinery.

     

Enzyme immunoassay of cyclic adenosine 3′,5′-monophosphate (AMP) using β-d-galactosidase as label

Succinyl cyclic adenosine monophosphate (AMP) was synthesized and coupled to β-D-galactosidase using 1-ethyl-3-(3-dimethylaminopropyl)carbodimide. An antiserum to cyclic AMP was raised in rabbits by immunization with a succinyl cyclic AMP-human serum albumin conjugate. An enzyme immunoassay of cyclic AMP was successfully performed via the competitive binding procedure using the succinyl cyclic AMP-enzyme conjugate and the antiserum to cyclic AMP. The sensitivity of this assay has been increased several hundredfold by prior 2′-O-succinylation of cyclic AMP as already described by Cailla et al. in the radioimmunoassay of cyclic nucleotides. The assay system makes it possible to ascertain values as low as 5 fmol of cyclic AMP/tube. Human plasma cyclic AMP could be accurately determined by this method without requiring a deproteinizing reagent as the first step of assay. The concentration of TCA-extracted cyclic AMP from various tissues of mice was determined by both enzyme immunoassay and radioimmunoassay. There was a good correlation between the values for cyclic AMP determined by the two methods (Y = 1.01X - 0.15, r = 0.996, n = 39). Succinylated nucleotides and nucleosides such as cyclic GMP, ATP, and AMP, adenosine, adenine, GTP, GDP, GMP, guanosine, and guanine had no effect on the immunoassay of cyclic AMP.     

Distribution and excretion of 2,3,6,2′,3′,6′- and 2,4,5,2′,4′,5′-hexachlorobiphenyl in senescent rats

The disposition of two symmetrical [¹⁴C]hexachlorobiphenyls (HCBs), 2,3,6,2′,3′,6′-HCB (236) and 2,4,5,2′,4′,5′-HCB (245), was studied in 24-month-old male Sprague-Dawley rats after iv treatment. Because body composition changes with age, complete dissections were performed on all rats to determine the size of the skin and adipose tissue depots. More than 50% of 236 was metabolized and excreted via the bile into the feces within 2 days. In contrast, 245 redistributed from the liver, muscle, and skin to adipose tissue where it accumulated without being metabolized. Only 2% of the total dose of 245 was excreted primarily in the feces within 21 days. The data obtained in this study were compared to results previously obtained from 2- to 3-month-old rats in this laboratory (Matthews and Tuey, 1980, Toxicol. Appl. Pharmacol.53, 377–388). Although the general pattern of HCB disposition did not change with age, i.e., metabolism and excretion of 236 versus persistence of 245, there were differences in the rates of elimination and in the tissue levels. There was enhanced metabolite retention in the muscle, skin, and adipose tissue of older animals which suggested an age-related decrease in tissue clearance. The large volume of adipose tissue in these older Sprague-Dawley rats could in part explain this observation. In general, there were few changes in decay rates from tissues or in biliary excretion. Age had a greater effect on the disposition of the persistent 245 than on the metabolizable 236. Thus, changes in body composition seemed to play a major role in age-related changes in the distribution and excretion of polychlorinated biphenyls.     

Stimulation of adenosine 3′,5′-monophosphate formation in guinea-pig cerebral cortical slices in a calcium free medium

Summary In guinea-pig cerebral cortical slices cyclic AMP concentrations increase during incubation with histamine+noradrenaline. After 10 min of incubation the levels of cyclic AMP start to decline. When calcium ions are omitted from the incubation medium, cyclic AMP levels do increase to a greater extent under the same conditions and do not drop during 30 min incubation. In the presence of calcium ions cyclic AMP synthesis can not be elicited by noradrenaline alone. In calcium-free Krebs-Ringer solution a pronounced effect of noradrenaline on cyclic AMP levels is observed. This effect of noradrenaline is shown to be mediated by a classical -type receptor. 5-Hydroxytryptamine, prostaglandin E ₁ and dopamine do not significantly enhance cyclic AMP formation in guinea-pig brain slices in either the presence in, or the absence of calcium ions from the incubation medium. Under depolarizing conditions of incubation the stimulatory effect of ouabain or 125 mM K⁺ is blocked in a calcium-free medium, while with the depolarizing agent veratridine no significant reduction of cyclic AMP formed during incubation in a calcium-free medium is obtained.     

Diethylstilbestrol potentiation of the dopamine-elicited formation of adenosine 3′,5′-monophosphate in incubated male rat hypothalamus

Summary Dopamine (DA) stimulates the cAMP-generating system in the male rat hypothalamus only to a very low extent (25% above control). Diethylstilbestrol (DES), a synthetic estrogen, was found to be extremely potent (a 4- and 16-fold stimulation at 20 M and 100 M, respectively). Addition of either one to an incubation medium containing varying concentrations of the other resulted in a synergistic response. The potentiation by 20 M DES of the effect elicited by 100 M DA was the most remarkable, namely, a 3-fold stimulation of the combined response. A 4- and 7.5-fold stimulation of cAMP accumulation was observed when adenosine (100 M) or adenosine (100 M)+DA (100 M) were present in the incubation medium. Theophylline (0.5 mM), an adenosine antagonist, could effectively reduce this effect, as did adenosine deaminase (10 g/ml). Clomiphene (50 M), an estrogen antagonist, exhibited a marked decrease in DES+DA-elicited cAMP formation. Pimozide (40 M) had the ability to significantly block the stimulatory effects of DES and DA.     

Characteristics of chick cerebral β-adrenoceptors assessed by cyclic adenosine 3′,5′ monophosphate formation and [3H]-propranolol binding

Summary Chick cerebral -adrenoceptors have been characterised by measurement of cyclic AMP accumulation in brain slices and assessment of the specific binding of [³H]-propranolol to cerebral membranes. The binding of [³H]-propranolol was inhibited by -adrenoceptor agonists and antagonists with affinities that correlated well with their ability to stimulate cyclic AMP formation or to antagonise the cyclic nucleotide accumulation induced by isoprenaline. The relative potencies of a number of drugs in several cerebral regions suggests that the receptors may be of the ₂ subtype. Regional distribution studies revealed the highest density of binding sites in the cerebellum and lowest in the optic lobes. However, the concentration of [³H]-propranolol that produced half-maximal specific binding was similar in all regions. Subcellular fractionation of cerebral hemisphere tissue demonstrated an enrichment of [³H]-propranolol binding sites in the synaptosomal and microsomal fractions. There are discrepancies between the topographical distribution of -adrenoceptor binding sites and the endogenous noradrenaline level in chick brain and between the number of binding sites and the intrinsic activity of -adrenoceptor mediated cyclic AMP formation.     

The influence of 8-Br 3′,5′-cyclic nucleotide analogs and of inhibitors of 3′,5′-cyclic nucleotide phosphodiesterase,on noradrenaline secretion and neuromuscular transmission in guinea-pig vas deferens

Summary The effects of 3,5-cyclic nucleotide phosphodiesterase (PDE) inhibitors and of 8-Br 3,5-cyclic nucleotide analogs on nerve-muscle transmission were studied in the guinea-pig vas deferens preincubated with ³H-noradrenaline.8-Br cyclic AMP and the PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX) and 3-propionyl-4-hydrazinopyrazolopyridine (SQ 20006) enhanced the secretion of ³H-NA evoked by transmural nerve stimulation. 8-Br cyclic GMP was without effect in this respect.The muscle contraction evoked by transmural nerve stimulation, high potassium or by application of exogenous noradrenaline was depressed by IBMX and SQ 20006. The contraction evoked by transmural nerve stimulation was enhanced by 8-Br cyclic AMP and depressed by 8-Br cyclic GMP.These findings suggest differential involvement of 3,5-adenosine- and guanosine-cyclic nucleotides in excitation-secretion-coupling in the noradrenergic sympathetic nerves, and in excitation-contraction-coupling in the smooth muscle, of guinea-pig vas deferens.     

Peroxisome proliferator-activated receptor-α activation attenuates 3-nitropropionic acid induced behavioral and biochemical alterations in rats: possible neuroprotective mechanisms

Peroxisome proliferators activated receptor is regarded as potential therapeutic targets to control various neurodegenerative disorders. However, none of the study has elucidated its effect in the treatment of Huntington's disease. We explored whether peroxisome proliferators activated receptor-α agonist may attenuate various behavioral and biochemical alterations induced by systemic administration of 3-nitropropionic acid (3-NP), an accepted experimental animal model of Huntington's disease phenotype. Intraperitoneal administration of 3-NP (20mg/kg., i.p.) for 4days in rats produced hypolocomotion, muscle incoordination, and cognitive dysfunction. Daily treatment with fenofibrate (100 or 200mg/kg., p.o.), 30min prior to 3-NP administration for a total of 4days, significantly improved the 3-NP induced motor and cognitive impairment. Biochemical analysis revealed that systemic 3-NP administration significantly increased oxidative and nitrosative stress (increase lipid peroxidation, protein carbonyls and nitrite level), lactate dehydrogenase activity whereas, decreased the activities of catalase, superoxide dismutase, reduced glutathione, and succinate dehydrogenase. Fenofibrate treatment significantly attenuated oxidative damage, cytokines and improved mitochondrial complexes enzyme activity in brain. In the present study, MK886, a selective inhibitor of peroxisome proliferators activated receptor-α was employed to elucidate the beneficial effect through either receptor dependent or receptor independent neuroprotective mechanisms. Administration of MK886 (1mg/kg, i.p.) prior to fenofibrate (200mg/kg, p.o.) abolished the effect of fenofibrate. The results showed that receptor dependent neuroprotective effects of fenofibrate in 3-NP administered rats provide a new evidence for a role of PPAR-α activation in neuroprotection that is attributed by modulating oxidative stress and inflammation.     

Influence of papaverine derivatives on phosphodiesterase activity,cyclic 3′,5′-AMP levels and relaxing effect on rabbit ileum

Summary The correlations between the relaxing effect of papaverine derivatives, inhibition of low K_m-phosphodiesterase (cAMP-PDE=EC 3.1.4.17) activity and cyclic 3,5-AMP (cAMP) levels in isolated rabbit ileum were investigated. There was a strong correlation between the relaxing effect, inhibition of PDE activity and cAMP content for eupaverine, ethylpapaverine and papaverine. Eupaverine was the most effective relaxing agent (I₅₀=7.5 M) and the most potent inhibitor of PDE activity (K_i=0.6 M), followed by ethylpapaverine (I₅₀=10 M); K_i=0.8 M) and papaverine (I₅₀=20 M; K_i=2 M). In contrast, there was a strong relaxing effect (I₅₀=6 M) but only slight inhibition of PDE activity (K_i=350 M) by tetrahydropapaveroline (THP). The adenylate cyclase stimulating effect of THP which was shown by others is most likely the reason for comparatively higher cAMP levels, which were found to be elevated about seven times over basal levels of 0.35 nmoles/g wet weight, and effective relaxation. Relaxation could be induced by exogenously added cAMP (I₅₀=45 M) and dibutyryl-cAMP (I₅₀=450 M). Our results support the assumption that smooth muscle relaxation in rabbit ileum is mediated by cAMP. Some of these observations have been published in abstract form (Schulz and Berndt, 1972).     

Effect of dopamine on adenosine 3′,5′-cyclic monophosphate levels in human platelets

1. The dopamine effect on intraplatelet adenosine 3′,5′-cyclic monophosphate (cAMP) levels was evaluated in healthy subjects.2. Dopamine levels over 30 nmol/l increased cAMP concentration in a concentration-dependent way.3. The platelet preincubation with propranolol (β-adrenoceptor antagonist) prevented the dopamine effect, whereas phentolamine (α-adrenoceptor antagonist) failed to modify the platelet response to this catecholamine. Dopamine receptor antagonist domperidone directly increased cAMP levels and it enhanced the dopamine effects.4. Our results indicate that dopamine effect is mainly mediated by β-adrenoceptor stimulation.     

Effects of methacholine,histamine and atropine on pulmonary guanosine-3′, 5′-monophosphate levels in hypersensitive mice

Summary Pulmonary levels of cGMP and cAMP in mice sensitized to methacholine and histamine with b. pertussis were examined to determine whether sensitization could be the result of an alteration in the metabolism of these cyclic nucleotides. The results presented show that in sensitized mice, methacholine raised cGMP to levels that were about double those produced without sensitization. In analogous experiments, histamine raised cGMP by approximately 100% in sensitized mice without producing significant increases in nonsensitized groups. Atropine completely blocked the cGMP rises produced by methacholine but did not eliminate those produced by histamine, thus indicating that cholinergic, but not the histaminergic elevation of cGMP involves activation of muscarinic receptors. The influence of pertussis on cAMP appeared to be opposite in direction from cGMP, i.e., a small but significant drop in cAMP levels was found following methacholine administration to sensitized, but not to nonsensitized mice. It was concluded that pertussis sensitization increases the responsiveness of the pulmonary guanylate cyclase-cGMP system to methacholine and histamine, and that the altered patterns of cGMP accumulation may contribute to the biochemical mechanism of sensitization.     

Disposition of 2′, 3′-dideoxyadenosine and 2′, 3′-dideoxyinosine in mice

Summary Mice were dosed with [³H]2,3-dideoxyadenosine ([³H]ddA) in three procedures: intravenously, intraperitoneally, and interperitoneally following a dose of 2 -deoxycoformycin (dCF). For mice dosed intravenously, the content of radioactivity in plasma and tissue samples were essentially constant after 30 min. Of the radioactivity in plasma and brain samples collected between 30 min and 24 hr, more than 94% was present as ³H₂O, indicating that most of the tritium from [³H]ddA had exchanged with water. No intact ddA was detected, and the deamination product, 2,3 -dideoxyinosine (ddI), was present only transiently. In the urine, the major radioactive material was [³H]ddI. Also detected were ³H₂O and small amounts of [³H]hypoxanthine and [³H]ddA. Following intraperitoneal doses to mice, levels of radioactivity in plasma, liver, and kidney increased to a maximum by 15–30 min after dosing but dropped to essentially constant levels thereafter, again indicating that the tritium had exchanged with water. At 5, 15, and 30 min after dosing, ddI was the major radioactive component in plasma. Only small amounts of ddA were present. When dCF was administered 24 hr prior to intraperitoneal [³H]ddA, levels of radioactivity in plasma, liver, and kidney reached a maximum at 30 to 60 min after dosing and decreased to essentially constant levels thereafter. The dCF transiently inhibited the deamination of ddA to ddI, since, in plasma, [³H]ddA was the main radioactive component at 5 and 15 min after dosing. Comparison of HPLC assays based on radioactivity detection and UV absorbance showed that they were equivalent for measuring ddA and ddI in samples derived from dosed mice. Therefore, exchange of tritium must have occurred at a metabolic step beyond ddI.For mice dosed intravenously and orally with unlabeled ddI, there was evidence of a saturated process. Nevertheless, for the high and low intravenous doses of ddI, the percent of dose excreted in the urine as unchanged drug was the same.     

Hyperlipidaemia induced by a high-cholesterol diet leads to the deterioration of guanosine-3′,5′-cyclic monophosphate/protein kinase G-dependent cardioprotection in rats

Background and purpose:

Hyperlipidaemia interferes with cardioprotective mechanisms, but the cause of this phenomenon is largely unknown, although hyperlipidaemia impairs the cardioprotective NO–cGMP system. However, it is not known if natriuretic peptide–cGMP–protein kinase G (PKG) signalling is affected by hyperlipidaemia. Therefore, we investigated the cardioprotective efficacy of cGMP-elevating agents in hearts from normal and hyperlipidaemic rats.

Experimental approach:

Male Wistar rats were rendered hyperlipidaemic by feeding with 2% cholesterol-enriched chow for 12 weeks. Hearts isolated from normal and hyperlipidaemic rats were perfused (Langendorff mode) and subjected to 30 min occlusion of the left main coronary artery, followed by 120 min reperfusion. 8-Br-cGMP (CG, 10 nM), B-type natriuretic peptide-32 (BNP, 10 nM), S-nitroso-N-acetyl-penicillamine (SNAP, 1 µM) were perfused from 10 min prior to coronary occlusion until the 15th min of reperfusion. Infarct size (% of ischaemic risk zone) was determined by triphenyltetrazolium staining.

Key results:

Treatment with CG, SNAP or BNP decreased infarct size significantly in normal hearts from its control value of 41.6 ± 2.9% to 15.5 ± 2.4%, 23.3 ± 3.0% and 25.3 ± 4.6%, respectively (P < 0.05). Protection by BNP was abolished by co-perfusion of PKG inhibitors KT5823 (600 nM) or Rp-8pCPT-PET-cGMPs (1 µM), confirming its PKG dependence. In hearts from hyperlipidaemic rats, CG, SNAP or BNP failed to decrease infarct size. Hyperlipidaemia did not alter basal myocardial PKG content, but decreased its activity as assessed by phosphorylation of cardiac troponin I.

Conclusions and implications:

This is the first demonstration that defects in the cardioprotective cGMP–PKG system could be a critical biochemical anomaly in hyperlipidaemia.     

Effect of some cyclooxygenase inhibitors on the increase in guanosine 3′:5′-cyclic monophosphate induced by NO-donors in human whole platelets

1. The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3′:5′-cyclic monophosphate (cyclic GMP) induced by nitric oxide-donor agents was tested in human whole platelets and in platelet crude homogenate.
2. In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide-donors (NO-donors) sodium nitroprusside (NaNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a dose-dependent way, its IC₅₀ being 13.7 μM and 15.8 μM, respectively.
3. Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO-donors in a dose-dependent way (IC₅₀=32.7 μM, NaNP and 25.0 μM, SNAP), while acetylsalicylic acid (up to 1000 μM) and diclofenac (up to 100 μM) were ineffective.
4. However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production.
5. Indomethacin (10 μM), indoprofen (30 μM), diclofenac (100 μM) and acetylsalicylic acid (1000 μM) showed a comparable efficacy in inhibiting platelet thromboxane B₂ (TXB₂) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO-donors was not mediated by inhibition of cyclooxygenase.
6. In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP.
7. The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO-donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen.

     

p38 MAPK mediates cardiovascular and behavioral responses induced by central IL-1β and footshock in conscious rats

INTRODUCTION Sharing characteristics of stress mediators[1], cen-tral interleukin-1β (IL-1β) is very important in cardio-vascular and behavioral responses to stressors[2-4]. Itcan induce similar hypertensive responses to stress viacardiovascular regulatory regions[5]. We observed pre-viously that central IL-1β mediated cardiovascular re-sponses to some stressful stimuli, such as conditionedfear stimuli and footshock[6]. Many stressful stimulimay increase IL-1β mRNA expression and …     

Differential involvement of 3′, 5′-cyclic adenosine monophosphate-dependent protein kinase in regulation of Fos and tyrosine hydroxylase expression in the heart after naloxone induced morphine withdrawal

We previously demonstrated that morphine withdrawal induced hyperactivity of the heart by the activation of noradrenergic pathways innervating the left and right ventricle, as evaluated by noradrenaline (NA) turnover and Fos expression. We investigated whether cAMP-dependent protein kinase (PKA) plays a role in this process by estimating changes in PKA immunoreactivity and the influence of inhibitor of PKA on Fos protein expression, tyrosine hydroxylase (TH) immunoreactivity levels and NA turnover in the left and right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg). When opioid withdrawal was precipitated, an increase in PKA immunoreactivity and phospho-CREB (cyclic AMP response element protein) levels were observed in the heart. Moreover, morphine withdrawal induces Fos expression, an enhancement of NA turnover and an increase in the total TH levels. When the selective PKA inhibitor HA-1004 was infused, concomitantly with morphine pellets, it diminished the increase in NA turnover and the total TH levels observed in morphine-withdrawn rats. However, this inhibitor neither modifies the morphine withdrawal induced Fos expression nor the increase of nonphosphorylated TH levels. The present findings indicate that an up-regulated PKA-dependent transduction pathway might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine withdrawal and suggest that Fos is not a target of PKA at heart levels.     

Increased responsiveness to adenosine 3′,5′-cyclic monophosphate-active agents during the immune response in vivo

Cytotoxic T lymphocytes (CTL) were generated in the spleen following immunization of C57BL/6 mice with allogeneic cells. The activity of such CTL was inhibited by agents that increased intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) levels. During the primary immune response, the susceptibility of CTL to inhibition by theophylline remained constant, but susceptibility to histamine, prostaglandin E₂ (PGE₂), dibutyryl cAMP, and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro-20-1724) increased significantly. The pattern of increased inhibition by histamine differed from that of the other agents. Increased cAMP responsiveness of CTL resulted from at least two distinct biochemical events, possibly including:

• 1.1) increased receptors and/or increased receptor-adenylate cyclase coupling (for histamine); and
• 2.2) increased efficiency of a post-cAMP pathway (for dibutyryl cAMP, PGE₂, and Ro-201724).

     

[3H]5′-N-ethylcarboxamide adenosine binds to both Ra and Ri adenosine receptors in rat striatum

Summary Adenosine analogs such as 5-N-ethylcarboxamide adenosine and N⁶-cyclohexyladenosine stimulate or inhibit adenosine cyclase activity in preparations of rat striatum depending on the assay conditions. N⁶-cyclohexyladenosine inhibits but does not stimulate adenosine cyclase activity in preparations of hippocampus. These findings suggest that the striatum contains both R _a (stimulatory) and R _i (inhibitory) adenosine receptors while the hippocampus contains only R _i receptors. We have previously shown that [³H]N⁶-cyclohexyladenosine binds to R _i receptors in rat hippocampus (Yeung and Green 1983). Comparisons of the characteristics of [³H]5-N-ethylcarboxamide adenosine and [³H]N⁶-cyclohexyladenosine binding to hippocampus show that [³H]5-N-ethylcarboxamide adenosine also binds to R _i receptors with high affinity. [³H]5-N-ethylcarboxamide adenosine binds to R _i receptors in the striatum and to a second site that is present in striatum but not hippocampus. High affinity binding of both ligands to R _i receptors can be blocked by treatments with N-ethylmaleimide that do not markedly affect [³H]5-N-ethylcarboxamide adenosine binding to the second site in the striatum. The pharmacological characteristics of the second site indicate that it is the R _a adenosine receptor.The abbreviations used are NEM N-ethylmaleimide - Gpp(NH)p 5-guanylylimidodiphosphate - NECA 5-N-ethylcarboxamide adenosine - l-PIA N⁶-(l-phenylisopropyl)adenosine - d-PIA N⁶-(d-phenylisopropyl) adenosine - DPX 1,3-diethyl-8-phenylxanthine