Preferential release of ATP and its extracellular catabolism as a source of adenosine upon high- but not low-frequency stimulation of rat hippocampal slices

The release of adenosine and ATP evoked by electrical field stimulation in rat hippocampal slices was investigated with the following two patterns of stimulation: (1) a brief, high-frequency burst stimulation (trains of stimuli at 100 Hz for 50 ms applied every 2 s for 1 min), to mimic a long-term potentiation (LTP) stimulation paradigm, and (2) a more prolonged (3 min) and low-frequency (5 Hz) train stimulation, to mimic a long-term depression (LTD) stimulation paradigm. The release of ATP was greater at a brief, high-frequency burst stimulation, whereas the release of [3H]adenosine was slightly greater at a more prolonged and low-frequency stimulation. To investigate the source of extracellular adenosine, the following two pharmacological tools were used: alpha, beta-methylene ADP (AOPCP), an inhibitor of ecto-5'-nucleotidase, to assess the contribution of the catabolism of released adenine nucleotides as a source of extracellular adenosine, and S-(4-nitrobenzyl)-6-thioinosine (NBTI), an inhibitor of adenosine transporters, to assess the contribution of the release of adenosine, as such, as a source of extracellular adenosine. At low-frequency stimulation, NBTI inhibited by nearly 50% the evoked outflow of [3H]-adenosine, whereas AOPCP inhibited [3H]adenosine outflow only marginally. In contrast, at high-frequency stimulation, AOPCP inhibited by 30% the evoked release of [3H]adenosine, whereas NBTI produced a 40% inhibition of [3H]adenosine outflow. At both frequencies, the kinetics of evoked [3H]adenosine outflow was affected in different manners by AOPCP and NBTI; NBTI mainly depressed the rate of evoked [3H]adenosine outflow, whereas AOPCP mainly inhibited the later phase of evoked [3H]adenosine accumulation. These results show that there is a simultaneous, but quantitatively different, release of ATP and adenosine from rat hippocampal slices stimulated at frequencies that can induce plasticity phenomena such as LTP (100 Hz) or LTD (5 Hz). The source of extracellular adenosine is also different according to the frequency of stimulation; i.e., at a brief, high-frequency stimulation there is a greater contribution of released adenine nucleotides for the formation of extracellular adenosine than at a low frequency with a more prolonged stimulation.     

Inhibition of protein synthesis induced by adenine nucleotides requires their metabolism into adenosine

Adverse reactions to food may be mediated by immunological or non immunological mechanisms. The term "food allergy" describes an event in which a definite immunopathological process can be demonstrated and a cause and effect relationship must be present. Symptoms and signs of food allergy may appear in any organ system, depending in part on the age of the subject and on the allergen involved. At present it is generally agreed that the only effective therapy for food allergy is strict elimination of the offending food antigen. Institution of a food elimination diet should be considered comparable to prescribing a medication, which carries along definite risk-benefit ratio. Consequently, appropriate diagnostic measures base on history, skin test, or radioallergosorbent test (Rast) and blind food challenges, must be utilized before implementing special diets. The allergist and other health care professionals must recognize the advantages of elimination diets (improvement of symptoms) as well as disadvantages (increase of the time required to purchase food and prepare meals, impossibility to eat at restaurants, at friends' houses or at school with consequent possible social isolation, nutritional disorders) and choose the most appropriate elimination diet.     

Hydroxylamine blocks adenosine A1 receptor-mediated inhibition of synaptic transmission in rat hippocampus

BACKGROUND AND PURPOSE: Stroke-induced hemiparesis involving the arm and hand results in regular, repeated overuse of the opposite hand and wrist. Because repetitive hand and wrist movement is a common cause of carpal tunnel syndrome (CTS), we examined the nonparetic upper limb in stroke patients for evidence of CTS. METHODS: We measured bilaterally sensory nerve conduction velocity (SNCV), motor nerve conduction velocity (MNCV), sensory nerve action potentials (SNAP) at the wrist, palm-to-wrist distal sensory latency (DSL), palm-to-wrist SNAP, compound motor action potentials (CMAP), and distal motor latency (DML) in stroke patients and control subjects. Controls were right-handed, >/=65 years old, lucid, independent in their activities of daily living, and had no disease known to cause CTS. Stroke patients were divided into a functioning hand group (n=61) and a disused hand group (n=71). All patients had hemiplegia. RESULTS: Tinel's sign was observed on the nonparetic side in 57.7% of patients with a disused hand and in 31.1% of those with a functioning hand. All electrophysiological indices were significantly more abnormal on the nonparetic side than on the hemiparetic side or in controls. Patients with a disused hand showed greater abnormality on the nonparetic side in SNCV, SNAP, palm-to-wrist DSL, DML, and CMAP than patients with a functioning hand. CONCLUSIONS: Overuse of the nonparetic hand and wrist of the nonparetic side may result in CTS in stroke patients, especially when the paretic hand is not functional. Wrist splinting or other prophylactic treatments beginning soon after stroke might help to prevent CTS.     

alpha-bungarotoxin- and methyllycaconitine-sensitive nicotinic receptors mediate fast synaptic transmission in interneurons of rat hippocampal slices

The role of nitric oxide (NO) in the long-term serotoninergic neurotoxicity induced by (+/-)3,4-methylenedioxymethamphetamine (MDMA) in rats was investigiated. Pretreatment with Nomega-nitro-L-arginine (L-NOARG) (10 mg kg-1), a nitric oxide synthase (NOS) inhibitor, partially protected against long-term serotonin (5-HT) depletion induced by MDMA (40 mg kg-1) in frontal cortex and parietal cortex, but not in other brain regions examined. Brain NOS activities in these two regions were significantly elevated at 6 h after MDMA administration. Moreover, L-NOARG pretreatment caused significant inhibition of brain NOS activity but did not affect the acute 5-HT and dopamine (DA) changes or the hyperthermia induced by MDMA. These results suggest that it is the NOS inhibitory properties of L-NOARG, rather than its effects on the acute monoamine changes or the hyperthermia induced by MDMA, that are responsible for the prevention of neurotoxicity. The regional differences on the protection of L-NOARG and on the activation of NOS by MDMA indicate the unequal role that NO may play in MDMA-induced neurotoxicity in different brain regions.     

Activation of hippocampal adenosine A3 receptors produces a desensitization of A1 receptor-mediated responses in rat hippocampus

The adenosine A3 receptor is expressed in brain, but the consequences of activation of this receptor on electrophysiological activity are unknown. We have characterized the actions of a selective adenosine A3 receptor agonist, 2-chloro-N6-(3-lodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), and a selective A3 receptor antagonist, 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191), in brain slices from rat hippocampus. In the CA1 region, activation of A3 receptors had no direct effects on synaptically evoked excitatory responses, long-term potentiation, or synaptic facilitation. However, activation of A3 receptors with Cl-IB-MECA antagonized the adenosine A1 receptor-mediated inhibition of excitatory neurotransmission. The effects of Cl-IB-MECA were blocked by pretreatment with MRS 1191, which by itself had no effect on A1 receptor-mediated responses. The presynaptic inhibitory effects of baclofen and carbachol, mediated via GABA(B) and muscarinic receptors, respectively, were unaffected by Cl-IB-MECA. The maximal response to adenosine was unchanged, suggesting that the primary effect of Cl-IB-MECA was to reduce the affinity of adenosine for the receptor rather than to uncouple it. Similar effects could be demonstrated after brief superfusion with high concentrations of adenosine itself. Under normal conditions, endogenous adenosine in brain is unlikely to affect the sensitivity of A1 receptors via this mechanism. However, when brain concentrations of adenosine are elevated (e.g., during hypoxia, ischemia, or seizures), activation of A3 receptors and subsequent heterologous desensitization of A1 receptors could occur, which might limit the cerebroprotective effects of adenosine under these conditions.     

Modulation of [3H]quinpirole binding to dopaminergic receptors by adenosine A2A receptors

Alteration of ligand binding to dopamine D2 receptors through activation of adenosine A2A receptors in rat striatal membranes has been studied by means of kinetic analysis. The binding of dopaminergic agonist [3H]quinpirole to rat striatal membranes was characterized by the constants Kd = 1.50+/-0.09 nM and Bmax = 115+/-2 fmol/mg of protein. The kinetic analyses revealed that the binding had at least two consecutive and kinetically distinguishable steps, the fast equilibrium of complex formation between receptor and agonist (KA = 5.9+/-1.7 nM), followed by a slow isomerization equilibrium (Ki = 0.06). Activation of adenosine A2A receptors by CGS 21680 caused enhancement of the rate [3H]quinpirole binding, altering mainly the formation of the receptor-ligand complexes (KA) as well as the isomerization rate of this complexes (ki), while the deisomerization rate (k[-i]) and the apparent dissociation rate remained unchanged.     

The structure and function of A1 and A2B adenosine receptors

Of the four G protein coupled adenosine receptor (AR) subtypes, the A1 is best suited for studies of reconstitution with G proteins. Recombinant A1 receptors extended with hexahistidine and FLAG have been purified to near homogeneity. In reconstitution assays using pure recombinant G protein subunits, the composition of the gamma subunit influences coupling to purified A1ARs. The least well characterized AR is the A2B. New data indicate that A(2B)ARs can trigger the degranulation of canine and human mast cell lines. Recombinant human A(2B)ARs are blocked by the anti-asthma drugs theophylline and enprofylline at concentrations that are used therapeutically to treat asthma. Although A(2B)ARs have long been known to stimulate adenylyl cyclase, they also can activate phospholipase C and mobilize Ca2+ by signaling through Gq/11. There is great potential for new therapies based on compounds that selectively target individual AR subtypes.     

Serotonin depresses excitatory synaptic transmission and depolarization-evoked Ca2+ influx in rat basolateral amygdala via 5-HT1A receptors

The actions of serotonin on rat basolateral amygdala neurons were studied with conventional intracellular recording techniques and fura-2 fluorimetric recordings. Bath application of 5-hydroxytryptamine (5-HT or serotonin) reversibly suppressed the excitatory postsynaptic potential in a concentration-dependent manner without affecting the resting membrane potential and neuronal input resistance. Extracellular Ba2+ or pertussis toxin pretreatment did not affect the depressing effect of 5-HT suggesting that it is not mediated through activation of Gi/o protein-coupled K+ conductance. The sensitivity of postsynaptic neurons to glutamate receptor agonist was unaltered by the 5-HT pretreatment. In addition, the magnitude of paired-pulse facilitation was increased in the presence of 5-HT indicating a presynaptic mode of action. The effect of 5-HT was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and was blocked by the selective 5-HT1A antagonist 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine oxadiazol-3-yl]methyl]phenyl]-methanesulphonamide. In contrast, the selective 5-HT2 receptor antagonist ketanserin failed to affect the action of 5-HT. The effects of 5-HT and 8-OH-DPAT on the high K+-induced increase in [Ca2+]i were studied in acutely dissociated basolateral amygdala neurons. High K+-induced increase in [Ca2+]i was blocked by Ca2+-free solution and Cd2+ suggesting that Ca2+ entry responsible for the depolarization-evoked increase in [Ca2+]i occurred through voltage-dependent Ca2+ channels. Application of 5-HT and 8-OH-DPAT reduced the K+-induced Ca2+ influx in a concentration-dependent manner. The effect of 5-HT was completely abolished in slices pretreated with Rp-cyclic adenosine 3',5'-monophosphothioate (Rp-cAMP), a regulatory site antagonist of protein kinase A, suggesting that 5-HT may act through a cAMP-dependent mechanism. Taken together, these results suggest that functional 5-HT1A receptors are present in the excitatory terminals and mediate the 5-HT inhibition of synaptic transmission in the amygdala.     

Differential expression of adenosine A1 and A2 receptors in preadipocytes and adipocytes

Multiple physiological functions have been described to be affected by adenosine in numerous cell types. A comparative study of the expression of adenosine receptors has been performed in preadipocytes and adipocytes from rat epididymal fat pad. The results show that, in agreement with its well known antilipolytic effect, adenosine induces a negative modulation of adenylate cyclase via the A1 receptor present in adipocytes. By contrast, the A2 receptor subtype, which is positively coupled to adenylate cyclase, is herein demonstrated to be only expressed in adipose precursor cells. This expression allows, in chemically defined medium, the adenosine analogue NECA, by means of its ability to elevate cAMP concentration, to potentiate differentiation. These findings emphasize the role that adenosine might play as a bimodal regulatory extracellular signal in adipose tissue development.     

Differential modulation of synaptic transmission by calcium chelators in young and aged hippocampal CA1 neurons: evidence for altered calcium homeostasis in aging

The effects of membrane-permeant Ca2+ chelators on field EPSPs (fEPSPs) were measured in the hippocampal CA1 region of brain slices from young (2-4 months) and old (24-27 months) Fischer 344 rats. BAPTA-AM depressed fEPSPs in young slices by up to 70% but enhanced fEPSPs by 30% in aged slices. EGTA-AM, with slower binding kinetics, did not affect fEPSPs from young slices but enhanced fEPSPs in aged slices. BAPTA derivatives with calcium dissociation constants (Kd) of 0.2-3.5 microM reduced or enhanced fEPSPs in young and aged slices, respectively, but 5',5'-dinitro BAPTA-AM (Kd of approximately 7000 microM) had no effect. Frequency facilitation of the fEPSPs occurred in young, but not in aged, slices, except when BAPTA-AM or EGTA-AM was perfused onto aged slices. The differential effects of BAPTA-AM in young and old slices were eliminated by perfusing with a low Ca2+-high Mg2+ saline or with the calcium blocker Co2+. These data suggest that intracellular Ca2+ regulation is altered and raised in aged neurons. Cell-permeant calcium buffers may be able to "ameliorate" deficits in synaptic transmission in the aged brain.     

Cross-modulation of synaptic plasticity by beta-adrenergic and 5-HT1A receptors in the rat basolateral amygdala

Neurotransmitter receptors are often colocalized in a neuron with other receptors, and activation of one receptor can either amplify or antagonize the response to a colocalized receptor. The aim of this study was to investigate the cross-regulation of synaptic transmission by beta-adrenergic and serotonin 1A (5-HT1A) receptors and to elucidate their underlying mechanisms. Stimulation of presynaptic beta-adrenergic receptors with isoproterenol (Iso) in the basolateral amygdala resulted in a long-lasting increase in synaptic transmission. This effect was mimicked by forskolin, an activator for adenylyl cyclase and a cAMP analog. In addition, the effect of forskolin was blocked by catalytic and regulatory site antagonists for cAMP-dependent protein kinase (PKA), indicating a PKA-mediated mechanism. Application of 5-HT depressed the synaptic transmission and blocked Iso- and forskolin-induced potentiation. The effect of 5-HT was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin and was blocked by the selective 5-HT1A antagonist 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine, indicating its mediation by 5-HT1A receptors. To determine the locus of interaction, Sp-cAMPS, a membrane-permeable activator of PKA, was applied, and the potentiation produced by Sp-cAMPS was completely blocked in slices pretreated with 5-HT. These results suggest that the interaction between the intracellular signaling pathways activated by 5-HT1A and beta-adrenergic receptors occurs at a step downstream from cAMP production.     

Opposite modulation of 4-aminopyridine and hypoxic hyperexcitability by A1 and A2 adenosine receptor ligands in rat hippocampal slices

A randomly selected, age-stratified sample of subjects 50 years of age and older, living in the Salford Health District area of Greater Manchester was drawn from the age-sex register of a four-doctor group practice and invited by post to enter a study of ptosis. Of 851 subjects approached, 499 (59%) replied. Of these, 99 refused to participate. The remaining 400 were visited at home and underwent a standardized protocol of ophthalmic history, and examination including a photograph of the eyes in the primary position. Forty-six (11.5%) of the subjects had ptosis and its prevalence increased with age. Ptosis was bilateral in 18 (39%) and unilateral in 28 (61%). In all but four cases, the ptosis was acquired. The cause was evident in 23 (50%), with 11 cases being due to mechanical ptosis and 12 to aponeurotic disinsertion secondary to a known pathology. A further 22 cases had primary aponeurotic disinsertion and there was one case of probable myasthenia gravis. The prevalence of pupillary diameter of 1 mm or less increased significantly with age.     

Binding of an adenosine A1 receptor agonist and adenosine A1 receptor antagonist to sheep pineal membranes

The pineal organ of vertebrates produces melatonin and adenosine. In lower vertebrates, adenosine modulates melatonin production. We report herein that 2-chloro-cyclopentyl-[3H]-adenosine ([3H]CCPA: adenosine A1 receptor agonist) and [3H]-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX: adenosine A1 receptor antagonist), bind specifically to sheep pineal membranes. Binding of [3H]CCPA reached equilibrium at 90 min and dissociation revealed the presence of two components. Saturation analysis suggested the presence of a single population of binding sites (Kd = 1.67 +/- 0.06 nM, Bmax = 2386 fmol/mg protein). Binding was sensitive to GTP and GTPgammaS. Binding of [3H]DPCPX reached equilibrium at 60 min and dissociation was monophasic. Saturation analysis revealed a single population of binding sites (Kd = 5.8 +/- 1.12 nM, Bmax = 1116 fmol/mg protein). The specificity of the [3H]-analogues used and the rank order potency of the competitors tested in the competition experiments suggested the presence of A1 receptors. Future investigations are necessary to elucidate the significance of the differences observed between the binding properties of the adenosine A1 receptor agonist and adenosine A1 receptor antagonist.     

Modulation of [3H]acetylcholine release from cultured amacrine-like neurons by adenosine A1 receptors

We have investigated the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultured chick amacrine-like neurons. The release of [3H]ACh evoked by 50 mM KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca2+ channels. Ligand binding studies using [3H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca2+-dependent release of [3H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3H]ACh release may be due to a direct inhibition of N-type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

The role of histamine H1, H2 and H3 receptors on enteric ascending synaptic transmission in the guinea pig ileum

The role of histamine H1-, H2- and H3-receptors was studied on neural transmission in ascending excitatory pathways of the guinea pig ileum. A two-compartment (oral and anal compartments) bath was used: ascending neural pathways were activated by electrical stimulation in the anal compartment and the resulting contraction of the circular muscle in the oral compartment was recorded. Drugs were applied in the anal compartment and each agonist was evaluated in the presence of the antagonists of the other two receptors. In the presence of cimetidine (10 microM) and thioperamide (1 microM), histamine (0.03-3 microM) depressed the nerve-mediated contractions (5-70% inhibition, P <.05-.01). The inhibitory effect of histamine was antagonized by mepyramine. At the higher concentrations (10 and 30 microM), histamine elicited contractions of the circular muscle in the oral compartment, and these were abolished by mepyramine (1 microM) and tetrodotoxin (0.6 microM). The H2 agonists dimaprit (30 and 100 microM) and amphamine (0.1-300 microM) produced small contractions of the circular muscle in the oral compartment. These contractile responses were abolished by tetrodotoxin (0.6 microM) and cimetidine (10 microM). The H3 agonist R-alpha-methylhistamine (0.001-1 microM) inhibited (2-58%, P <.05) the nerve-mediated contractions. This inhibitory effect was antagonized by the H3 antagonist thioperamide. These results indicate that 1) histamine, acting at H1 receptors, at lower concentrations depresses synaptic transmission, although at higher concentrations activates the enteric excitatory ascending pathway; 2) activation of H2 receptors by H2 agonists stimulates the enteric excitatory ascending pathways and 3) activation of H3 receptors inhibits synaptic transmission.     

Activation of metabotropic glutamate receptors induce differential effects on synaptic transmission in the dentate gyrus and CA1 of the hippocampus in the anaesthetized rat

Activation of ACPD-sensitive metabotropic receptors induced differential effects on synaptic transmission and the induction of LTP in CA1 and the dentate gyrus of the hippocampus i.c.v. injections of (1.S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced enduring potentiation of the fEPSP in CA1, which occluded tetanically induced LTP. In contrast, ACPD induced a dose-dependent biphasic effect on the fEPSP in the dentate gyrus, consisting of an initial short lasting potentiation, followed by enduring depression of the response, and blockade of LTP. These two effects are likely to be mediated by two different classes of the receptor as in the dentate gyrus the selective class I agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG) induced sustained potentiation of the fEPSP, whereas the mixed mGluR2 agonist-mGluR1 antagonist, (S)-4-carboxy-3-hydrophenylglycine((S)-4C3H-PG) induced only depression. Increasing the concentration of calcium directly in the dentate gyrus prior to, and in conjunction with, injections of ACPD induced sustained potentiation rather than depression. The differential effects indicate that the second messenger cascades the subtypes of receptors are linked with, mediate different forms of synaptic plasticity within the hippocampus and have important implications for their role in learning.     

Prolonged enhancement and depression of synaptic transmission in CA1 pyramidal neurons induced by transient forebrain ischemia in vivo

Evoked postsynaptic potentials of CA1 pyramidal neurons in rat hippocampus were studied during 48 h after severe ischemic insult using in vivo intracellular recording and staining techniques. Postischemic CA1 neurons displayed one of three distinct response patterns following contralateral commissural stimulation. At early recirculation times (0-12 h) approximately 50% of neurons exhibited, in addition to the initial excitatory postsynaptic potential, a late depolarizing postsynaptic potential lasting for more than 100 ms. Application of dizocilpine maleate reduced the amplitude of late depolarizing postsynaptic potential by 60%. Other CA1 neurons recorded in this interval failed to develop late depolarizing postsynaptic potentials but showed a modest blunting of initial excitatory postsynaptic potentials (non-late depolarizing postsynaptic potential neuron). The proportion of recorded neurons with late depolarizing postsynaptic potential characteristics increased to more than 70% during 13-24 h after reperfusion. Beyond 24 h reperfusion, approximately 20% of CA neurons exhibited very small excitatory postsynaptic potentials even with maximal stimulus intensity. The slope of the initial excitatory postsynaptic potentials in late depolarizing postsynaptic potential neurons increased to approximately 150% of control values up to 12 h after reperfusion indicating a prolonged enhancement of synaptic transmission. In contrast, the slope of the initial excitatory postsynaptic potentials in non-late depolarizing postsynaptic potential neurons decreased to less than 50% of preischemic values up to 24 h after reperfusion indicating a prolonged depression of synaptic transmission. More late depolarizing postsynaptic potential neurons were located in the medial portion of CA1 zone where neurons are more vulnerable to ischemia whereas more non-late depolarizing postsynaptic potential neurons were located in the lateral portion of CA1 zone where neurons are more resistant to ischemia. The result from the present study suggests that late depolarizing postsynaptic potential and small excitatory postsynaptic potential neurons may be irreversibly injured while non-late depolarizing postsynaptic potential neurons may be those that survive the ischemic insult. Alterations of synaptic transmission may be associated with the pathogenesis of postischemic neuronal injury.     

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

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

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.     

Pharmacological characterization of a simple behavioral response mediated selectively by central adenosine A1 receptors, using in vivo and in vitro techniques

Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of [125I]salmon (s) CT(1-32) ([125I]sCT(1-32)) was 56 fmol/mg protein, and of [125I]chicken (ch) [Tyr36]PTHrP(1-36) amide ([125I]chPTHrP(1-36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1-32) (EC50 0.05 nM) and 3-fold by chPTHrP(1-36) (EC50 1.3 nM). Receptor autoradiography revealed specific binding of [125I]sCT(1-32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1-32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1-36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.