Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry implications for drug addiction, sleep and pain.

Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of l-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A)receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent.

Decrease of D2 receptor binding but increase in D2-stimulated G-protein activation, dopamine transporter binding and behavioural sensitization in brains of mice treated with a chronic escalating dose 'binge' cocaine administration paradigm.

Understanding the neurobiology of the transition from initial drug use to excessive drug use has been a challenge in drug addiction. We examined the effect of chronic 'binge' escalating dose cocaine administration, which mimics human compulsive drug use, on behavioural responses and the dopaminergic system of mice and compared it with a chronic steady dose (3 x 15 mg/kg/day) 'binge' cocaine administration paradigm. Male C57BL/6J mice were injected with saline or cocaine in an escalating dose paradigm for 14 days. Locomotor and stereotypy activity were measured and quantitative autoradiographic mapping of D(1) and D(2) receptors, dopamine transporters and D(2)-stimulated [(35)S]GTPgammaS binding was performed in the brains of mice treated with this escalating and steady dose paradigm. An initial sensitization to the locomotor effects of cocaine followed by a dose-dependent increase in the duration of the locomotor effect of cocaine was observed in the escalating but not the steady dose paradigm. Sensitization to the stereotypy effect of cocaine and an increase in cocaine-induced stereotypy score was observed from 3 x 20 to 3 x 25 mg/kg/day cocaine. There was a significant decrease in D(2) receptor density, but an increase in D(2)-stimulated G-protein activity and dopamine transporter density in the striatum of cocaine-treated mice, which was not observed in our steady dose paradigm. Our results document that chronic 'binge' escalating dose cocaine treatment triggers profound behavioural and neurochemical changes in the dopaminergic system, which might underlie the transition from drug use to compulsive drug use associated with addiction, which is a process of escalation.

Comparative binding of mu and delta selective ligands in whole brain and pons/medulla homogenates from rat: affinity profiles of fentanyl derivatives.

The comparative binding characteristics of the mu opioid receptor selective ligand [3H]-[D-Ala2-MePhe4-glyol5]enkephalin [( 3H]-DAGO) and of the delta receptor ligand [3H]-[D-Pen2, D-Pen5]enkephalin[( 3H]-DPDPE) have been studied in homogenates of both whole brain and of pons/medulla regions from the rat. The receptor affinities of five 4-anilinopiperidine drugs (fentanyl derivatives) and of morphine have been determined by inhibition studies, using [3H]-DAGO and [3H]-DPDPE as markers of the mu and delta opioid binding sites, respectively. The concentration of delta opioid sites in pons/medulla was found to be approximately one third that of mu sites. The concentrations of both mu and delta sites in whole brain were similar to that of mu sites in pons/medulla. The rank order of affinities of the unlabelled drugs was dissimilar at the mu and delta sites. The most potent fentanyl derivatives exhibited negligible preference for the mu or delta sites, in contrast to the least potent compound, alfentanil which showed an extremely high mu-site selectivity.

Behavioural profiles of putative 5-hydroxytryptamine receptor agonists and antagonists in developing rats.

The effects of a variety of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on behaviour in 5- and 20-day old rat pups have been investigated. Increased locomotion and head-weaving responses were induced in both age groups by 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin; 5-HT1A agonist); 5-MeODMT (5-methoxy-N,N-dimethyltryptamine; 5-HT1) and RU 24969 (5-methoxy-3(1,2,3,6-tetrahydropyrindin-4-yl)-1H-indole; 5-HT1B/5-5HT1A). The putative 5-HT1A-agonist LY165163 (1-2-(4-aminophenyl)ethyl 4-(3-trifluoromethylphenyl)piperazine) also produced hyperactivity in the developing pups. In contrast, locomotion was not affected by buspirone (5-HT1A); mCPP (1-(3-chlorophenyl)piperazine; 5-HT1B/5-HT1C) and DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; 5-HT2) though buspirone produced a small increase in head-weaving at 5- and 20-days. The full 5-HT syndrome was induced in older animals (but not neonates) by both 8-OH-DPAT and 5-MeODMT. Large doses of buspirone, mCPP and DOI also produced signs of reciprocal forepaw treading and flattened body posture at 20-days. In addition, mCPP induced grooming and stereotyped mouthing, while DOI increased sniffing behaviour in the young rats. Catecholaminergic mechanisms were implicated in the head-weaving and locomotor responses to 8-OH-DPAT and RU 24969, following experiments with a number of monoamine receptor antagonists. Preliminary findings with (-)-pindolol, which was high affinity for 5-HT1-receptors, suggested that this subtype of receptor may play a role in hyperlocomotion induced by RU 24969.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of 8-OH-DPAT induced spontaneous tailflicks as an in vivo model of 5-HT1A function in young rats.

Spontaneous tailflicks, measured as elevation of the tail above the body axis in restrained animals, have been studied in both adult and young rats. The selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), induced dose-related increases in spontaneous tailflicks in adult (> 60 days) male rats. These responses were antagonised by the 5-HT1A antagonists (-)-propranolol and (-)-pindolol. 8-OH-DPAT (1 mg/kg) induced tailflicks could be observed in 30, 25 and 20 day old male rats and were also antagonised by (-)-propranolol and (-)-pindolol although drug-induced rotation of the 20 and 25 day old animals hindered assessment. At 14 and 10 days, 8-OH-DPAT (1 mg/kg) produced Straub tail responses which precluded the observation of tailflicks. Lower doses of 8-OH-DPAT, which did not elicit Straub tail in these younger animals also failed to produce tailflicks. Thus 8-OH-DPAT induced spontaneous tailflicks reflect in vivo activation of 5-HT1A receptors in adult and 30 day old male rats but are inappropriate for the study of 5-HT1A mediated behaviour in younger animals.

Nociceptin/orphanin FQ knockout mice display up-regulation of the opioid receptor-like 1 receptor and alterations in opioid receptor expression in the brain.

The opioid receptor-like 1 receptor is a novel member of the opioid receptor family and its endogenous peptide ligand has been termed nociceptin and orphanin FQ. Activation of the opioid receptor-like 1 receptor by nociceptin/orphanin FQ in vivo produces hyperalgesia when this peptide is given supraspinally but analgesia at the spinal level. Nociceptin/orphanin FQ also reverses stress-induced analgesia, suggesting that the peptide has anti-opioid properties. Nociceptin/orphanin FQ knockout mice show alterations in pain sensitivity and stress responses and display increased morphine dependence, suggesting an interaction of the nociceptin/orphanin FQ system with classical opioid receptor function. To determine if the behavioural phenotype of nociceptin/orphanin FQ knockout mice reflects changes in either opioid receptor-like 1 or classical opioid receptor expression, we have carried out quantitative autoradiography of the opioid receptor-like 1, mu-, delta- and kappa-opioid receptors in the brains of these animals. Receptor density was measured on coronal sections from wild-type, heterozygous and homozygous mice using [(3)H]nociceptin, [(3)H][D-Ala(2)-N-methyl-Phe(4)-Gly(5) ol] enkephalin, [(3)H]deltorphin-I, or [(3)H](-)-N-methyl-N-[7-(1-pyrrodinyl)-1-oxospiro[4,5]dec-8-yl]-4-benzofuranacetamide to label opioid receptor-like 1, mu-, delta- and kappa-receptors, respectively. A region-specific up-regulation of the opioid receptor-like 1 receptor (up to 135%) was seen in brains from homozygous mice. Mu-Receptors also showed significant differences between genotypes whilst changes in delta- and kappa- receptors were minor. In conclusion the region-specific up-regulation of the opioid receptor-like 1 receptor indicates a tonic role for nociceptin/orphanin FQ in some brain structures and may suggest the peptide regulates the receptor expression in these regions. The changes in the opioid receptor-like 1 receptor may relate to the anxiogenic phenotype of these animals but the observed change in mu-receptors does not correlate with altered morphine responses.

Region selective up-regulation of micro-, delta- and kappa-opioid receptors but not opioid receptor-like 1 receptors in the brains of enkephalin and dynorphin knockout mice.

The role of endogenous opioid peptides and receptors has recently been investigated using knockout mice. Although the affinities of opioid peptides for opioid receptors has been known for many years there is still some uncertainty over which receptor is the endogenous target for each peptide. To address this issue we have studied using quantitative autoradiography the levels of all four opioid receptor subtypes (micro, delta, kappa and opioid receptor-like 1 [ORL1]) in brains sectioned from enkephalin and dynorphin knockouts, as well as from double knockouts. Because receptor up-regulation has been observed when its cognate ligand-peptide is genetically ablated, regional changes in receptor binding in knockout mice may reflect areas where the peptide ligand is tonically active at its receptor or played a role in receptor regulation. In addition, the study aimed to correlate previously observed behaviour in these animals with receptor modulation. Marked region-specific up-regulation of the micro, delta, and kappa opioid receptors but not ORL1 receptors was observed in proenkephalin and prodynorphin knockouts. In proenkephalin knockouts this was most pronounced for the micro- and delta-receptor and in prodynorphin knockouts for the kappa-receptor. Combinatorial double knockouts did not show any changes in addition to those observed in single knockouts. The largest changes were observed in limbic regions and our results suggest that proenkephalin peptides are tonically active at micro and delta-receptors predominantly in these areas. Prodynorphin peptides appear to regulate mostly the kappa-receptor but they are also modulators of micro- and delta-receptors.

Autoradiographic mapping of the opioid receptor-like 1 (ORL1) receptor in the brains of mu-, delta- or kappa-opioid receptor knockout mice.

The opioid receptor-like 1 (ORL1) receptor shares a high degree of sequence homology with the classical mu-, delta- and kappa-opioid receptors and a functional mutual opposition between these receptors has been suggested. To further address this possible interaction we have used mu-, delta- and kappa-opioid receptor knockout mice to determine autoradiographically if there are any changes in the number or distribution of the ORL1 receptor, labelled with [(3)H]nociceptin, in the brains of mice deficient in each of the opioid receptors. An up-regulation of ORL1 expression was observed across all brain regions in delta-knockouts with cortical regions typically showing a 15-30% increase in binding that was most marked in heterozygous mice. In contrast, ORL1 receptor expression was down-regulated in virtually all brain structures in heterozygous kappa-knockouts although the magnitude of this change was not as great as for the delta-knockouts. No significant alterations in ORL1 receptor expression were observed across brain regions in mu-receptor knockout mice and there were no qualitative differences in ORL1 receptor expression in any groups. These data suggest there are interactions between the ORL1 system and the classical opioid receptors and that the interactions are receptor-specific. The greater differences observed in heterozygous mice suggest that these interactions might be most relevant when there is only partial loss of receptor function.

Perinatal lead exposure alters the development of delta- but not mu-opioid receptors in rat brain.

1. Low level lead exposure has been shown to impair the development of opioid peptide levels in the brain, and to impair antinociceptive responses to opioid drugs. We have now studied the effects of lead exposure on the development of opioid receptors using ligand binding studies. 2. The ontogenesis of mu- and delta-opioid binding sites was studied using rat whole brain membranes and [3H]-[D-Ala2MePhe4-Gly-ol]enkephalin and [3H]-[D-Pen2,D-Pen5]enkephalin as binding ligands. Rats were exposed to lead during development by addition of lead acetate (at 100-1000 p.p.m.) to the maternal drinking water from conception to postnatal day 14. 3. Perinatal lead exposure had no significant effects on the binding affinity (KD) or binding capacity (Bmax) for the mu-opioid receptor measured at postnatal days 10, 21 and 30. Lead exposure (at 1000 p.p.m.) increased the KD for the delta-opioid receptor at postnatal days 15, 21, 35 and 50 but had no effect on the binding capacity. No indications of overt toxicity were observed and blood lead levels were in the ranges considered to represent subclinical lead toxicity in man. 4. The lack of effect of lead on mu-receptor binding contrasts with previously described impairment of antinociceptive effects of mu-agonists suggesting that the toxicity is not manifested at the mu-binding site. However, the delta-opioid receptor appears to be more sensitive to lead exposure and the persistent changes in delta-site affinity after cessation of lead exposure suggest irreversible damage in the production of the receptor protein.

Developmental responses to opioids reveals a lack of effect on stress-induced corticosterone levels in neonatal rats.

The neonate has an unusual capacity for survival and the possibility exists that mechanisms for controlling stress responses may differ in the developing animal. In adults both endogenous and exogenous opioids can modulate the corticosterone responses to stress. We have studied this effect in neonatal rats and found that opioid modulation is absent in early postnatal development. Neonatal rats of either sex were injected with morphine (5-50 mg kg-1), fentanyl (10-100 micrograms kg-1), buprenorphine (0.1-30 mg kg-1) or naloxone (0.1-10 mg kg-1) and plasma corticosterone measured fluorimetrically 15 or 20 min later. In addition naloxone reversibility studies (1 mg kg-1, co-administered) were carried out for the opioid agonists. In adult rats, elevations in plasma corticosterone caused by injection stress were potentiated by morphine, fentanyl and buprenorphine. In neonates, though injection stress-induced rises in plasma corticosterone were absent at 10 days, elevations were observed at 21 days and later. However, significant potentiation of this corticosterone response by fentanyl was absent at 21 days and at later ages (30 and 40 days) for morphine and buprenorphine. The potentiating effect of all three agonists did not become fully effective until day 45. In addition, in animals acclimatized to injection stress by 7 day injection pretreatment, fentanyl did not significantly alter corticosterone levels in 30 day old neonates. High doses of naloxone (10 mg kg-1) significantly increased the corticosterone response to injection stress in adult rats but this effect was absent in 30 day old animals. A dose of naloxone (I mg kg-') which had no significant effect on the corticosterone response inhibited the effects of morphine, fentanyl and buprenorphine in 45 day old and adult rats. 5 This late development of opioid action is unusual in comparison with the maturation of endogenous peptides, receptors and antinociceptive responses and suggests that alternative mechanisms may be involved in stress-control in the neonate.

Effect of delayed weaning on opioid receptor control of swim stress-induced antinociception in the developing rat.

1. The opioid type of swim-stress induced antinociception (SIA) is mediated via mu-sites in preweanling rats and predominantly by delta-sites in postweanling animals. We have studied the effect of delay of weaning on the receptor transition of this behaviour in the developing rat. 2. Litters were weaned normally at day 21 or allowed to remain with their mothers until assessment of swim SIA. Animals were stressed by warm water (20 degrees C) swimming for 3 min periods and antinociception assessed by the tail immersion test (50 degrees C). 3. Naloxone (10 mg kg-1) partially reversed swim SIA in both 25 day old weaned and non-weaned rats. 4. Naltrindole (1 mg kg-1) partially reversed swim SIA in 25 day old weaned rats but had no effect in non-weaned animals. Naltrindole (5 mg kg-1) completely abolished swim SIA in weaned rats but was without effect in non-weaned groups. Antinociceptive responses to the mu-agonist, alfentanil (60 micrograms kg-1) were unaffected by naltrindole at 1 mg kg-1 but were partially reversed at 5 mg kg-1. 5. In 30 day old non-weaned rats, naltrindole (5 mg kg-1) abolished the swim SIA. 6. In conclusion, transition from mu to delta-receptor control of swim SIA in rat pups can be delayed by between 5 and 10 days by delay of weaning. The environmental stimulus of weaning can activate opioid receptor subtype operation of biological responses in the developing animal.

Perinatal lead exposure impairs opioid but not non-opioid stress-induced antinociception in developing rats.

1. The development of opioid systems has been shown to be sensitive to perinatal exposure to lead. We have studied the effects of such exposure on opioid and non-opioid mediated stress-induced antinociception in developing rats. 2. Lead was administered in the maternal drinking water from conception to postnatal day 14 at 300 and 1000 p.p.m. Twenty and 25 day old rats were subjected to swimming stress and antinociception measured using the tail immersion test. 3. A 3 min swim-stress induced an opioid-mediated antinociceptive response in 20 day old rats which was attenuated by 300 p.p.m. lead and by 1000 p.p.m. lead treatment in a dose-related manner. A 10 min swim-stress induced a non-opioid mediated antinociceptive response in 25 day old rats which was not antagonised by 300 or 1000 p.p.m. lead. 4. Naloxone antagonised the residual antinociception observed in 20 day old animals treated with 300 p.p.m. lead and had no effect on antinociception in control or lead-treated 25 day old rats. 5. Using a lead exposure model considered to represent subclinical lead toxicity in man, it was shown that perinatal lead exposure disrupts opioid but not non-opioid mediated stress antinociception.

Swim-stress-induced antinociception in young rats.

1. Opioid and non-opioid mechanisms have been implicated in the phenomenon of stress-induced antinociception in adult rodents. We have studied stress-induced antinociception in developing rats and characterized differences in the neurochemical basis of this effect in pre- and post-weanling animals. 2. Twenty and 25 day old rats were stressed using warm water (20 degrees C) swimming for 3 or 10 min periods and antinociception was assessed by the tail immersion test (50 degrees C). 3. A 3 min swim in 20 and 25 day old rats produced marked antinociception which was blocked by naloxone, Mr 1452, 16-methyl cyprenorphine and levallorphan but not Mr 1453 or N-methyl levallorphan. The delta-opioid receptor antagonist ICI 174,864 attenuated stress-induced antinociception in 25 day old rats but was without effect in 20 day old animals. 4. A 10 min swim in 25 day old rats produced antinociception which was non-opioid in nature. In contrast, antinociception was not observed in 20 day old rats after a 10 min swim-stress. 5. Pretreatment of animals with dexamethasone blocked 3 min swim-stress antinociception in 20 and 25 day old animals but had no effect on antinociception induced by a 10 min swim. 6. Swim-stress-induced antinociception can be observed in young rats and dissociated into opioid and non-opioid types dependent on the duration of swimming stress. The non-opioid type appears to develop more slowly and cannot be observed in preweanling rats. The opioid type is a predominantly mu-receptor phenomenon in preweanling animals but delta-receptor components are observable in postweanling rats.

Different effects of di-isopropylfluorophosphate on the entry of opioids into mouse brain.

Di-isopropylfluorophosphate (DFP) potentiates the antinociceptive activity of alfentanil but has no effect on the activity of morphine or fentanyl. We have studied the effect of DFP on the distribution of these three opioids in the brain. Distribution studies were carried out using 3H-labelled opioids administered subcutaneously to mice. Animals were killed at times of peak antinociceptive activity and 3H-opioid measured in plasma and in eight brain regions. DFP pretreatment (1 mg kg-1) caused a significant increase in the brain:plasma ratio of alfentanil in all brain regions but had no effect on brain:plasma ratios for morphine or fentanyl. The enhanced entry of alfentanil into the brain of DFP-treated mice probably accounts for the increased antinociception observed with this opioid. This drug interaction appears to be opioid specific.

Antagonism of swim-stress-induced antinociception by the delta-opioid receptor antagonist naltrindole in adult and young rats.

1. The availability of the non-peptide delta-opioid receptor antagonist naltrindole has provided the possibility for in vivo studies on the function of delta-opioid receptors. We have studied the effects of naltrindole on swim-stress-induced antinociception in adult and neonatal rats. 2. Adult, 25 and 20 day old rats were stressed by warm water (20 degrees C) swimming for 3 min periods and antinociception was assessed by the tail immersion test (50 degrees C). 3. Naltrindole (0.5 and 1 mg kg-1) antagonized swim-stress-induced antinociception in adult and 25 day old rats but in 20 day old rats naltrindole (1 mg kg-1) was without effect. 4. Antinociception induced by the highly mu-opioid receptor selective agonist alfentanil was completely antagonized by naloxone (1 mg kg-1) but virtually unaffected by naltrindole (1 mg kg-1). 5. Neither naloxone nor naltrindole (1 mg kg-1) antagonized swim-stress-induced rises in plasma corticosterone in adult rats at the time of peak antinociception. 6. In conclusion, naltrindole shows in vivo antagonism of opioid-mediated responses. Swim-stress-induced antinociception is mediated through the delta-opioid receptor in 25 day old and adult rats and through the mu-opioid site in 20 day old animals.

Effects of the delta-opioid receptor antagonist naltrindole on antinociceptive responses to selective delta-agonists in post-weanling rats.

1. Antagonism, by the selective delta-opioid receptor antagonist naltrindole, of the antinociceptive effects of [D-Pen2, D-Pen5] enkephalin (DPDPE), [D-Ser2, Leu5, Thr6] enkephalin (DSLET) and D-Ala2 deltorphin I (DELT I) has been studied in 25 day old rats. 2. Antinociception was measured by the 50 degrees C tail immersion test following i.p. administration of agonists and/or antagonists. 3. Dose-related antinociception was observed with DPDPE, DSLET and DELT I and ED75 doses were computed (0.66 mg kg-1, 0.65 mg kg-1, 0.032 mg kg-1 respectively) and used for antagonism studies. 4. Naltrindole (0.01 mg kg-1) significantly attenuated the antinociceptive effects of DPDPE and DSLET with 0.1 mg kg-1 producing complete reversal of the effects of the ED75 dose. In contrast, naltrindole at 0.01 and 0.1 mg kg-1 did not alter antinociceptive responses to DELT I. Naltrindole at 1 mg kg-1 significantly attenuated DELT I antinociception. 5. Naloxone (1 mg kg-1) produced equivalent degrees of antagonism of the antinociceptive effects of DPDPE, DSLET and DELT I. ICI 174,864 (1 mg kg-1) also antagonized antinociception with a differential degree of attenuation (DSLET > DPDPE > DELT I). 6. Naltrindole (1 mg kg-1) had no effect on the antinociception induced by the selective mu-agonist alfentanil (60 micrograms kg-1). Naltrindole, naloxone or ICI 174,864 had no effect on nociceptive latencies. 7. The differential antagonism by naltrindole of the effects of three selective delta-agonists suggests delta-receptor heterogeneity.Further, the lower sensitivity of response to DELT I suggests that this agent may exert its antinociceptive effects at a different 6 receptor subtype from DPDPE or DSLET.

Characterization of P1-purinoceptors on rat duodenum and urinary bladder.

1. The P1-purinoceptors mediating relaxation of the rat duodenum and inhibition of contraction of the rat urinary bladder were characterized by use of adenosine and its analogues 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyladenosine (CPA) and 2-p-((carboxyethyl)phenethylamino)-5'- carboxamidoadenosine (CGS 21680), as well as the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable analogue of adenosine 5'-triphosphate (ATP), adenylyl 5'-(beta,gamma-methylene)diphosphonate (AMPPCP), was also used as previous work had indicated that it has a direct action on some P1 receptors in addition to its P2-purinoceptor activity. 2. In the rat duodenum, the order of potency of the adenosine agonists was NECA greater than or equal to CPA greater than AMPPCP = adenosine greater than CGS 21680, and DPCPX antagonized CPA and AMPPCP at a concentration of 1 nM whereas equivalent antagonism of NECA and adenosine required a concentration of 1 microM. This suggests the presence of a mixture of A1 and A2 receptors in this tissue, with CPA and AMPPCP acting on the A1 and NECA and adenosine acting on the A2 receptors. 3. In the rat bladder, the order of potency of the adenosine agonists for inhibition of carbachol-induced contractions was NECA much greater than adenosine greater than CPA = CGS 21680, and a concentration of DPCPX of 1 microM was required to antagonize responses to NECA and adenosine. This suggests the presence of A2 receptors in this tissue. ATP and AMPPCP each caused contractions which were not enhanced by DPCPX (1 microM) which suggests that in this tissue AMPPCP was acting only via P2 receptors and had no P1 agonist activity. That AMPPCP was active on the A1 receptors in the duodenum but inactive on the A2 receptors in the bladder implies that it has selectivity for the A1 subtype.4. That CGS 21680, which has been reported to bind selectively to the high affinity A2a subclass of A2 receptors, had a very low potency on the A2 receptors in the duodenum and in the bladder suggests that these receptors are of the low affinity A2b subclass.

Differential development of adenosine A1 and A2b receptors in the rat duodenum.

1. The development of the adenosine A1 and A2b receptors inducing relaxation of the rat duodenum was studied by use of a combination of functional and radioligand binding assays on rats aged between 5 and 30 days and compared with results previously found in adult rat duodenum. 2. 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) bound with high affinity to a single site in duodenum preparations from rats aged 20, 25 and 30 days. At 10 and 15 days there was no detectable specific binding of [3H]-DPCPX. 3. The affinity (KD) of the binding site for [3H]-DPCPX was similar in membrane preparations from 20, 25 and 30 day old animals (1.58-2.27 nM), but the density (Bmax) of binding sites was found to increase up to 25 days where peak levels (72.0 +/- 9.5 fmol mg-1 protein) were observed and then decline at 30 days (45.5 +/- 2.9 fmol mg-1 protein) to levels commensurate with those previously determined in the adult rat duodenum. 4. In duodenum from 10 day old rats no responses to N6-cyclopentyladenosine (CPA, 1 nM-10 microM) were observed, at 15 days the duodenum responded to the highest concentration of CPA (3 microM) only, and at 20-30 days concentration-related responses were observed, with the potency of CPA increasing with an increase in age. DPCPX (10 nM) abolished the responses to CPA except at the highest concentration of CPA (3 microM) where the response was markedly attenuated, suggesting the presence of an A1, receptor. 5. In rat duodenum from animals of all ages (5-30 days), concentration-related responses to 5'-N-ethylcarboxamidoadenosine (NECA) were observed. The potency of NECA remained constant with an increase in age, whereas the maximum relaxation response increased from 20% at 5 days to 110% at 25 and 30 days. In the presence of 1 microM DPCPX a right-ward shift in the concentration-response curve to NECA was observed at all ages. In the presence of 10 nM DPCPX, the response to NECA was unaffected in the duodenum from animals aged 10 and 15 days. However, in duodenum from animals aged 20-30 days the concentration-response curve to NECA was shifted to the right suggesting that there is an A1 component to the action of NECA at these ages. Schild analysis of the effects of increasing concentrations of DPCPX versus NECA on the duodenum from 25 day old animals generated a slope of 0.62 suggesting that NECA acts at A1 and A2b receptors as in the adult. 6. The A2b-selective analogue, 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) (10 nM-10 microM) was without effect on the carbachol-contracted duodenum from 15 day old rats and the duodenum from 25 day old rats responded to the highest concentration of CGS 21680 only, suggesting that the A2 receptors here, as in the adult, are not of the A2a subtype. The adenosine antagonist, 8-phenyltheophylline (8-PT) (10 microM), abolished the inhibitory effects of NECA (100 nM-100 microM) on 10, 15 and 25 day old rat duodenum indicating that the responses to NECA were not mediated via an adenosine A3 receptor. 7. These results show that adenosine A1 receptors in rat duodenum are present and functionally viable from day 20 onwards and that the density of A1 receptors varies with age, increasing up to day 25 and then declining at day 30 to a density commensurate with that found in the adult. The responses to CPA, mediated via the A1 receptor, increase with age in a similar fashion. In contrast however, the response to NECA was evident from day 5, the earliest age studied, and from days 5-15 NECA acted via the A2b receptor subtype. However, from day 20 onwards NECA acted at a mixed population of A1 and A2b receptors. These results demonstrate the differential development of the A1 and the A2b receptors in the rat duodenum.

Different effects of current strength on inhibitory responses of the mouse vas deferens to methionine- and leucine-enkephalin.

The inhibitory potency of methionine (Met)-enkephalin on the field-stimulated mouse vas deferens was greatly increased by a reduction in current strength whilst that of leucine (Leu)-enkephalin increased only slightly. All currents were submaximal and all muscle twitches were neuronally evoked. These results suggest that inhibitory effects of Met- and Leu-enkephalin in the mouse vas deferens are not commonly mediated and provide a rapid method for ascertaining heterogeneity of enkephalin extracts.

The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine to adenosine A1 receptors in rat smooth muscle preparations.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2. [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 +/- 0.47 nM), colon muscularis mucosae (0.84 +/- 0.15 nM), duodenum (1.59 +/- 0.18 nM) and vasa deferentia (0.93 +/- 0.17 nM). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 +/- 4 fmol mg-1 protein), muscularis mucosae (43 +/- 3.5 fmol mg-1 protein) and vasa deferentia (43.3 +/- 12.2 fmol mg-1 protein), but in the longitudinal muscle 6-7 fold more binding sites (295 +/- 70 fmol mg-1 protein) were identified. 3. Inhibition studies using DPCPX (0.1-100 nM), N6-cyclopentyladenosine (CPA) (0.1-100 nM), 5'-N-ethylcarboxamidoadenosine (NECA) (2 nM-10 microM) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nM-1 microM) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nM), demonstrated an order of potency of displacement in all tissues of DPCPX > or = CPA > R-PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia. Two site analysis revealed that the agonists bind to both a high and low affinity state of the receptor.4. The existence of Al binding sites in the rat vasa deferentia, colon muscularis mucosae and duodenum, and their absence in the urinary bladder, is consistent with previous functional studies.However, in contrast to the findings of the [3H]-DPCPX binding assay, no functional response mediated by adenosine Al receptors could be detected by measuring contractile or relaxant responses to CPA in the colon longitudinal muscle. The functional significance of the binding sites in this tissue has therefore yet to be determined.