enkephalin--ala(2)-mephe(4)-gly(5)- and 1-3-dipropyl-8-cyclopentylxanthine

Methylxanthines like caffeine and theophylline have long been used to treat apnea of prematurity. Despite their success in stimulating neonatal breathing, their mechanism of action remains poorly understood. Methylxanthines can act as both non-specific adenosine receptor antagonists and inhibitors of cAMP-dependent phosphodiesterases, sarcoplasmic/endoplasmic reticulum calcium ATPases or receptor-coupled anion channels, depending on the dose used. Though there is evidence for methylxanthine action at the level of the carotid body, the consensus is that methylxanthines stimulate the respiratory centers of the brainstem. Here we used the in situ neonatal rat working heart-brainstem preparation and the ex vivo neonatal rat carotid body preparation to test the hypothesis that methylxanthines act at the level of the carotid body. We conclude that although the neonatal carotid body has active adenosine receptors, the effects of methylxanthine therapy are likely mediated centrally, predominantly via inhibition of cAMP-dependent phosphodiesterase-4.

Topics: Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Animals; Animals, Newborn; Apnea; Brain Stem; Carotid Body; Central Nervous System Stimulants; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Phrenic Nerve; Quinazolines; Rats, Sprague-Dawley; Respiration; Theobromine; Theophylline; Tissue Culture Techniques; Triazoles; Xanthines

The endocannabinoid system is emerging as an integral component in central and peripheral regulation of feeding and energy balance. Our investigation analyzed behavioral roles for cannabinoid mechanisms of the pontine parabrachial nucleus (PBN) in modulating intake of presumably palatable foods containing fat and/or sugar. The PBN serves to gate neurotransmission associated with, but not limited to, the gustatory properties of food. Immunofluorescence and in vitro [(35)S]GTPgammaS autoradiography of rat tissue sections containing the PBN revealed the presence of cannabinoid receptors and their functional capability to couple to their G-proteins after incubation with the endocannabinoid 2-arachidonoyl glycerol (2-AG). The selective cannabinoid 1 receptor (CB(1)R) antagonist AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] prevented the response, demonstrating CB(1)R mediation of 2-AG-induced coupling. Microinfusions of 2-AG into the PBN in behaving rats robustly stimulated feeding of pellets high in content of fat and sucrose (HFS), pure sucrose, and pure fat (Crisco), during the first 30 min after infusion. In contrast, 2-AG failed to increase consumption of standard chow, even when the feeding regimen was manipulated to match baseline intakes of HFS. Orexigenic responses to 2-AG were attenuated by AM251, again indicating CB(1)R mediation of 2-AG actions. Furthermore, responses were regionally specific, because 2-AG failed to alter intake when infused into sites approximately 500 mum caudal to infusions that successfully stimulated feeding. Our data suggest that hedonically positive sensory properties of food enable endocannabinoids at PBN CB(1)Rs to initiate increases in eating, and, more generally, these pathways may serve a larger role in brain functions controlling behavioral responses for natural reward.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Autoradiography; Behavior, Animal; Cannabinoid Receptor Modulators; Conditioning, Operant; Eating; Endocannabinoids; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Food Preferences; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Male; Narcotic Antagonists; Peptides; Piperidines; Pons; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Sulfur Isotopes; Time Factors; Xanthines

Relapse is the most serious limitation of effective medical treatment of opiate addiction. Opiate-related behaviors appear to be modulated by cannabinoid CB1 receptors (CB1) through poorly understood cross-talk mechanisms. Opiate and CB1 receptors are coexpressed in the nucleus accumbens (NAc) and dorsal striatum. These regions also have the highest density of adenosine A2a receptors (A2a) in the brain. We have been investigating the postsynaptic signaling mechanisms of mu-opiate receptors (MORs) and CB1 receptors in primary NAc/striatal neurons. In this article, we present evidence that MOR and CB1 act synergistically on cAMP/PKA signaling in NAc/striatal neurons. In addition, we find that synergy requires adenosine and A2a. Importantly, an A2a antagonist administered either directly into the NAc or indirectly by i.p. injection eliminates heroin-induced reinstatement in rats trained to self-administer heroin, a model of human craving and relapse. These findings suggest that A2a antagonists might be effective therapeutic agents in the management of abstinent heroin addicts.

Topics: Adenosine A2 Receptor Antagonists; Analgesics, Opioid; Animals; Arachidonic Acids; Behavior, Animal; Corpus Striatum; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Activation; Gene Expression Regulation; Heroin Dependence; Humans; Male; Neurons; Nucleus Accumbens; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Self Administration; Signal Transduction; Theobromine; Xanthines

The role of the adenosine A1 receptor in nociception was assessed using mice lacking the A1 receptor (A1R-/-) and in rats. Under normal conditions, the A1R-/- mice exhibited moderate heat hyperalgesia in comparison to the wild-type mice (A1R+/+). The mechanical and cold sensitivity were unchanged. The antinociceptive effect of morphine given intrathecally (i.t.), but not systemically, was reduced in A1R-/- mice and this reduction in the spinal effect of morphine was not associated with a decrease in binding of the mu-opioid ligand DAMGO in the spinal cord. A1R-/- mice also exhibited hypersensitivity to heat, but not mechanical stimuli, after localized inflammation induced by carrageenan. In mice with photochemically induced partial sciatic nerve injury, the neuropathic pain-like behavioral response to heat or cold stimulation were significantly increased in the A1R-/-mice. Peripheral nerve injury did not change the level of adenosine A1 receptor in the dorsal spinal cord in rats and i.t. administration of R-PIA effectively alleviated pain-like behaviors after partial nerve injury in rats and in C57/BL/6 mice. Taken together, these data suggest that the adenosine A1 receptor plays a physiological role in inhibiting nociceptive input at the spinal level in mice. The C-fiber input mediating noxious heat is inhibited more than other inputs. A1 receptors also contribute to the antinociceptive effect of spinal morphine. Selective A1 receptor agonists may be tested clinically as analgesics, particularly under conditions of neuropathic pain.

Topics: Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Analgesics, Opioid; Analysis of Variance; Animals; Behavior, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Functional Laterality; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Nociceptors; Pain Measurement; Photochemistry; Protein Binding; Radioligand Assay; Rats; Reaction Time; Receptor, Adenosine A1; Sciatica; Statistics, Nonparametric; Time Factors; Xanthines

The purpose of this study was to investigate the cellular basis of the synergistic anti-nociceptive interaction between adenosine and opioids reported for spinal cord in vivo. Patch clamp recordings from rat substantia gelatinosa neurons in vitro were used to assess whether adenosine receptor antagonists impact upon mu-opioid receptor (MOR)-mediated inhibition of glutamatergic synaptic transmission. The MOR agonist DAMGO inhibited evoked EPSCs and this inhibition was partly reversed by DPCPX, an A1 receptor (A1R) antagonist. The A2a receptor antagonist, ZM241385 had mixed effects on DAMGO-mediated inhibition, producing either a further inhibition or a reversal of the inhibition. These data show that activation of A1R as a secondary consequence of MOR-activation and putative adenosine release will potentiate opioid synaptic inhibition of nociceptive circuitry.

Topics: Adenosine; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Postsynaptic Potentials; Neural Inhibition; Neurons; Organ Culture Techniques; Patch-Clamp Techniques; Rats; Receptors, Opioid, mu; Receptors, Purinergic P1; Substantia Gelatinosa; Synapses; Triazines; Triazoles; Xanthines

Delta-opioid receptor (DOPr) activation fails to produce cellular physiological responses in many brain regions, including the periaqueductal gray (PAG), despite neural expression of high densities of the receptor. Previous histochemical studies have demonstrated that a variety of stimuli, including chronic morphine treatment, induce the translocation of DOPr from intracellular pools to the surface membrane of CNS neurons. PAG neurons in slices taken from untreated mice exhibited mu-opioid receptor (MOPr) but not DOPr-mediated presynaptic inhibition of GABAergic synaptic currents. In contrast, after 5-6 d of chronic morphine treatment, DOPr stimulation inhibited synaptic GABA release onto most neurons. Shorter exposure to morphine in vitro (upto 4 h) or in vivo (18 h) did not induce functional DOPr responses. DOPr-mediated presynaptic inhibition could not be induced in slices from untreated animals by increasing synaptic activity in vitro using high extracellular potassium concentrations or activation of protein kinase A. Induction of functional DOPr signaling by chronic morphine required MOPr expression, because no DOPr receptor responses were observed in MOPr knock-out mice. DOPr agonists also had no effect on miniature IPSCs in beta-arrestin-2 knock-out mice after chronic morphine. These results suggest that induction of DOPr-mediated actions in PAG by chronic morphine requires prolonged MOPr stimulation and expression of beta-arrestin-2.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Arrestins; beta-Arrestin 2; beta-Arrestins; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Excitatory Amino Acid Antagonists; G Protein-Coupled Inwardly-Rectifying Potassium Channels; gamma-Aminobutyric Acid; Glycine Agents; In Vitro Techniques; Isoquinolines; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Narcotic Antagonists; Narcotics; Neurons; Oligopeptides; Patch-Clamp Techniques; Periaqueductal Gray; Protein Kinase Inhibitors; Receptors, Opioid, delta; Receptors, Opioid, mu; Strychnine; Sulfonamides; Synaptic Transmission; Time Factors; Xanthines

There is evidence showing that the opioid and adenosine systems play an important role in cocaine addiction; fewer studies have examined their roles in cocaine withdrawal. To determine whether cocaine and/or chronic withdrawal from cocaine alters the specific components of the opioid and adenosine systems, we carried out quantitative autoradiographic mapping of mu-opioid, A1 and A2A adenosine receptors in the brains of rats treated with an escalating dose "binge" cocaine administration paradigm and of rats chronically withdrawn from cocaine. Male Fischer rats were injected with saline or cocaine (15 x 3 mg/kg/day for 4 days, 20 x 3 mg/kg/day for 4 days, 25 x 3 mg/kg/day for 4 days and 30 x 3 mg/kg/day for 2 days) at 1-h intervals for 14 days. Similarly treated rats were withdrawn from that paradigm for 14 days. A significant increase in [(3)H]DAMGO binding to mu-receptors was detected in the frontal and cingulate cortex, as well as in the caudate putamen, of long-term withdrawn rats after an escalating dose "binge" cocaine administration paradigm and in chronic cocaine-treated rats. No significant cocaine-induced change was found in A1 or A2A receptor binding in any region analyzed. These results reconfirm that mu-opioid (MOP) receptors undergo upregulation in response to chronic escalating dose "binge" cocaine administration. This upregulation was shown for the first time to persist at least 14 days into withdrawal after escalating "binge" cocaine.

Topics: Adenosine; Analgesics, Opioid; Animals; Autoradiography; Behavior, Animal; Brain; Brain Mapping; Cocaine; Dopamine Uptake Inhibitors; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Phenethylamines; Protein Binding; Purinergic P1 Receptor Antagonists; Rats; Rats, Inbred F344; Receptors, Opioid, mu; Receptors, Purinergic P1; Substance Withdrawal Syndrome; Tritium; Up-Regulation; Xanthines

Opioids delivered to the pons inhibit REM sleep, whereas pontine administration of adenosine enhances REM sleep. In other brain areas opioids and adenosine interact to produce antinociception. Adenosine A1 receptors and mu opioid receptors each activate Gi/Go proteins. This study tested the hypothesis that combined treatment with the adenosine A1 receptor agonist SPA and the mu opioid agonist DAMGO would enhance G protein activation to a greater level than produced by either agonist alone. G protein activation was quantified in seven brainstem regions regulating sleep and nociception. This study also tested the hypothesis that G protein activation caused by SPA would be concentration dependent and blocked by the adenosine A1 receptor antagonist DPCPX.. Activation of G proteins was assessed autoradiographically by agonist stimulation of [35S]GTPgammaS binding in slide-mounted sections of rat brainstem. G protein activation was quantified in nCi/g tissue for pontine reticular formation, dorsal raphe, ventrolateral and dorsomedial periaqueductal gray, and laterodorsal and pedunculopontine tegmental nuclei.. N/A.. Combined treatment with SPA and DAMGO caused a partially additive increase in G protein activation that was significantly (p<0.01) greater than G protein activation caused by either agonist alone. Treatment with SPA alone caused a concentration dependent (p<0.001) increase in [35S]GTPgammaS binding that was blocked by DPCPX.. Agonist activation of adenosine A1 receptors stimulates G proteins in brainstem nuclei regulating sleep and nociception. In these same nuclei, G protein activation by combined treatment with DAMGO and SPA was partially additive, suggesting that mu opioid and adenosine A1 receptors activate some common G protein pools.

Topics: Analgesics, Opioid; Animals; Autoradiography; Binding, Competitive; Drug Therapy, Combination; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Male; Mesencephalon; Nociceptors; Pons; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Receptors, Purinergic P1; Reticular Formation; Sleep, REM; Xanthines

1. An eye-cup preparation in anaesthetized rabbits was used to examine opioid modulation of acetylcholine (ACh) release from cholinergic neurones in the retina. 2. The mu-opioid receptor agonist, [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO), when applied locally to the retina at concentrations between 1-30 microM significantly increased the light-evoked release of ACh. The effect of DAMGO was completely blocked by the selective mu-receptor antagonist CTOP but the kappa-receptor antagonist nor-binaltorphimine (norBNI) did not affect the action of DAMGO on ACh release indicating that the opioid produced its effect by activation of mu-receptors (the rabbit retina has negligible delta-receptors). 3. Blockade with bicuculline and strychnine of GABAergic and glycinergic inputs to the cholinergic neurones did not affect the action of DAMGO on ACh release. Also DAMGO did not reduce the potassium-evoked release of either GABA or glycine from rat isolated retinas. 4. Exposure of the rabbit retina to a combination of an A1-adenosine receptor antagonist, 8-cyclopentyl-1,3 dipropylxanthine (DPCPX), and adenosine deaminase did not affect the enhancing action of DAMGO on the light-evoked release of ACh. 5. When the retina in the rabbit eye-cup was exposed to kainate, the release of ACh was increased by approximately three times the resting release. In the presence of DAMGO the kainate-evoked release of ACh was enhanced by 44%. 6. These experiments show that activation of mu-opioid receptors by DAMGO increases the release of ACh elicited by physiological stimulation (flickering light). Since we could find no evidence thatDAMGO reduces inhibitory inputs to the cholinergic neurones, it seems that the enhancing action ofDAMGO on the light-evoked release of ACh involves a direct excitatory effect rather than disinhibition.This conclusion is supported by the enhancing action of DAMGO on the kainate-evoked release of ACh because kainate is thought to act directly on the cholinergic neurones.

Topics: Acetylcholine; Animals; Bicuculline; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; gamma-Aminobutyric Acid; Glycine; Kainic Acid; Male; Rabbits; Rats; Receptors, Opioid, mu; Retina; Strychnine; Xanthines