guanosine-diphosphate and 1-3-dipropyl-8-cyclopentylxanthine

Adenosine produces cardiovascular depressor effects in various brain regions. However, the cellular mechanisms underlying these effects remain unclear. The pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) play an important role in regulating arterial blood pressure and sympathetic outflow through projections to the spinal cord and brainstem. In this study, we performed whole-cell patch-clamp recordings on retrogradely labeled PVN neurons projecting to the intermediolateral cell column of the spinal cord in rats. Adenosine (10-100 microM) decreased the firing activity in a concentration-dependent manner, with a marked hyperpolarization in 12 of 26 neurons tested. Blockade of A(1) receptors with the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or intracellular dialysis of guanosine 5'-O-(2-thodiphosphate) eliminated the inhibitory effect of adenosine on labeled PVN neurons. Immunocytochemical labeling revealed that A(1) receptors were expressed on spinally projecting PVN neurons. Also, blocking ATP-dependent K(+) (K(ATP)) channels with 100 microM glibenclamide or 200 microM tolbutamide, but not the G protein-coupled inwardly rectifying K(+) channels blocker tertiapin-Q, abolished the inhibitory effect of adenosine on the firing activity of PVN neurons. Furthermore, glibenclamide or tolbutamide significantly decreased the adenosine-induced outward currents in labeled neurons. The reversal potential of adenosine-induced currents was close to the K(+) equilibrium potential. In addition, adenosine decreased the frequency of both spontaneous and miniature glutamatergic excitatory post-synaptic currents and GABAergic inhibitory post-synaptic currents in labeled neurons, and these effects were also blocked by 8-cyclopentyl-1,3-dipropylxanthine. Collectively, our findings suggest that adenosine inhibits the excitability of PVN pre-sympathetic neurons through A(1) receptor-mediated opening of K(ATP) channels.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adenosine; Analgesics; Animals; Bicuculline; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glyburide; Guanosine Diphosphate; In Vitro Techniques; KATP Channels; Male; Membrane Potentials; Neurons; Paraventricular Hypothalamic Nucleus; Patch-Clamp Techniques; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Sodium Channel Blockers; Tetrodotoxin; Thionucleotides; Tolbutamide; Triazines; Triazoles; Xanthines

The receptor-stimulated accumulation of [35S]GTPgammaS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autoradiographic visualization of [35S]GTPgammaS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specific (NSB), basal and agonist-stimulated [35S]GTPgammaS binding were measured, using methadone, 5-carboxamidotryptamine and epinephrine for mu-opiate receptors, 5-HT(1A) receptors and alpha(2)-adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist-stimulated [35S]GTPgammaS binding was more easily observed. Further optimization using glycylglycine buffer found increased signal-to-noise ratio with inclusion of dithiothrietol, increased [35S]GTPgammaS incubation time (2-4 h) and guanosine 5'-diphosphate (GDP) preincubation (20-30 min), and use of [35S]GTPgammaS at 0.1 nM. Improved sensitivity was due to decreased NSB and basal [35S]GTPgammaS binding and agonist-stimulated binding were similarly affected for each receptor system. The assay conditions described should extend the use of agonist-stimulated [35S]GTPgammaS autoradiography to receptors, which produce low levels of [35S]GTPgammaS binding and to the measurement of changes in receptor-G protein coupling.

Topics: Adrenergic Agonists; Adrenergic alpha-2 Receptor Agonists; Animals; Autoradiography; Binding, Competitive; Brain; Buffers; Dithiothreitol; Dose-Response Relationship, Drug; Epinephrine; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Magnesium; Methadone; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; Receptors, Serotonin, 5-HT1; Sensitivity and Specificity; Serotonin; Serotonin Receptor Agonists; Sodium Chloride; Sulfur Radioisotopes; Time Factors; Xanthines

The influence of the receptor-G protein coupling state and the guanine nucleotide ligation state of the G protein on the binding mechanism of A(1) adenosine receptor ligands has been investigated in [(3)H]-1,3-dipropyl-8-cyclopentylxanthine ([(3)H]-DPCPX) binding studies in rat brain membranes. Thermodynamic parameters of binding of A(1) adenosine receptor ligands of different intrinsic activities were determined in the absence or presence of GDP and compared to the binding mechanism after receptor-G protein uncoupling. In agreement with previous studies, it was found that xanthine and non-xanthine antagonists showed an enthalpy- or enthalpy- and entropy-driven binding mechanism under all conditions. In contrast to antagonists, the binding mechanism of agonists was strongly affected by the G protein coupling state or the absence or presence of guanine nucleotides. Binding of full and partial agonists to the high-affinity state of the A(1) receptor was entropy-driven in the absence of GDP, and a good correlation between intrinsic activities and the contribution of entropy was observed. In the absence of GDP, binding of full and partial agonists and antagonists to the high affinity state of the receptor was thermodynamically discriminated. In contrast, no such discrimination was found in the presence of GDP. The binding mechanism of agonists to the low-affinity state of the receptor was identical to that of antagonists only after uncoupling of the receptor from G proteins by pretreatment with N-ethylmaleimide or guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS). These results indicate the existence of two thermodynamically distinct high- and low-affinity states of the A(1) adenosine receptor.

Topics: Animals; Brain; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Diphosphate; In Vitro Techniques; Ligands; Prosencephalon; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Receptors, Purinergic P1; Thermodynamics; Xanthines

The adenosine receptor agonist N(6)-cyclohexyl[(3)H]adenosine ([(3)H]CHA) was used to identify and pharmacologically characterize adenosine A1 receptors in brown trout (Salmo trutta) brain. In membranes prepared from trout whole brain, the A1 receptor agonist [(3)H]CHA bound saturably, reversibly and with high affinity (K(d)=0. 69+/-0.04 nM; B(max)=0.624+/-0.012 pmol/mg protein) to a single class of binding sites. In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [(3)H]CHA from high-affinity binding sites with the rank order of potency characteristic for an adenosine A1 receptors. A1 receptor density appeared not age-related (from 3 months until 4 years), and was similar in different brain areas. The specific binding was inhibited by guanosine 5'-triphosphate (IC(50)=0.778+/-0.067 microM). GTP (5 microM) induced a low affinity state of A1 receptors. In superfused trout cerebral synaptosomes, 30 mM K(+) stimulated the release of glutamate in a calcium dependent manner. Glutamate-evoked release was dose-dependently reduced by CHA, and the inhibition was reversed by the A1 antagonist 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, 30 mM K(+) as well as 1 mM glutamate stimulated the release of adenosine in a Ca(2+)-independent manner and tetrodotoxin insensitive. These findings show that in trout brain adenosine A1 receptors are present which are involved in the modulation of glutamate transmitter release. Moreover, the stimulation of adenosine release by K(+) depolarisation or glutamate support the hypothesis that, as in mammalian brain, a cross-talk between adenosine and glutamate systems exists also in trout brain.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding, Competitive; Brain; Cell Membrane; Glutamic Acid; Guanosine Diphosphate; Kinetics; Neuroprotective Agents; Radioligand Assay; Receptors, Purinergic P1; Synaptosomes; Theophylline; Tritium; Trout; Xanthines

Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H1, H2, and H3. We have used guanosine 5'-(gamma-[35S]thio)triphosphate (GTPgamma[35S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A1 receptor-dependent GTPgamma[35S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 microM), a selective A1 receptor antagonist. Histamine elicited dose-dependent increments in GTPgamma[35S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H3 binding sites and was faithfully mimicked by N(alpha)-methylhistamine, (R)-alpha-methylhistamine, and immepip, three H3-selective agonists. In all regions examined, the GTPgamma[35S] signal was reversed with thioperamide and clobenpropit, two potent H3-selective antagonists, whereas mepyramine, a specific H1 antagonist, and cimetidine, a prototypic H2 antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPgamma[35S] autoradiography selectively detects H3 receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPgamma[35S] autoradiography preferentially reveals responses to G(i/o)-coupled receptors, our data indicate that most, if not all, central H3 binding sites represent functional receptors coupling to G(i/o), the inhibitory class of G proteins. Besides allowing more detailed studies on H3 receptor signaling within anatomically restricted regions of the CNS, GTPgamma[35S] autoradiography offers a novel approach for functional in vitro screening of H3 ligands.

Topics: Adenosine; Animals; Autoradiography; Basal Ganglia; Brain Chemistry; Cerebral Cortex; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Histamine; Histamine Antagonists; Histamine H1 Antagonists; Male; Piperidines; Protein Binding; Pyrilamine; Rats; Rats, Wistar; Receptors, Histamine H3; Signal Transduction; Substantia Nigra; Sulfur Radioisotopes; Xanthines

In a previous study we showed that in vivo treatment with pertussis toxin could inhibit some, but not all, effects of adenosine in the rat hippocampus. In this study we investigated the effect of pertussis toxin on the binding of adenosine analogues to A1 receptors in rat brain. Intraventricular injection of pertussis toxin (10 micrograms into the lateral ventricle) did not affect A1 receptor binding in any brain region studied, as evaluated by autoradiography. In vitro treatment of brain sections (10 microns) with pertussis toxin for 5 h, under conditions when greater than 80% of the G proteins were ADP ribosylated, did not alter radioligand binding to adenosine A1 receptors. GTP (10 microM) virtually abolished the high-affinity agonist binding to the A1 receptor. On the other hand, in solubilized cortical membrane preparations, pertussis toxin pretreatment induced a complete shift of the A1 receptors to the low-affinity state. This suggests that the ability of pertussis toxin to affect G proteins coupled to A1 receptors in brain depends not only on the distribution of the toxin but also on the configuration of receptors and G proteins.

Topics: Adenosine; Adenosine Diphosphate Ribose; Animals; Autoradiography; Brain; Ethylmaleimide; Guanosine Diphosphate; Magnesium Chloride; Male; Pertussis Toxin; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Purinergic; Solubility; Virulence Factors, Bordetella; Xanthines