Adenosine binding sites of rat pheochromocytoma PC 12 cell membranes: partial characterization and solubilization

Rat pheochromocytoma PC 12 cell membranes were shown to possess A2-like adenosine binding sites as assessed by using 5'-N-ethylcarboxamide[3H]adenosine [( 3H]NECA). Specific [3H]NECA binding to PC 12 cell membrane at 0 degrees C was saturable and showed a monophasic saturation profile. In contrast, [3H]NECA binding to PC 12 cell membrane at 30 degrees C exhibited a biphasic profile suggesting the presence of two specific binding site. The rank order of potency for inhibition of [3H]NECA binding at 0 degrees C was NECA greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine much greater than phenylisopropyladenosine. These adenosine binding sites were solubilized with sodium cholate and the solubilized portion retained the same ligand binding characteristics as those of the membrane-bound form. Gel filtration experiments indicated an apparent Stokes radius of 6.7 nm for these adenosine binding sites/detergent complexes.     

Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2 Dopamine Receptors in the A1 Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor-G protein-adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet-activating protein, IAP) and apomorphine on A1 adenosine agonist (-)N6-R-[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 +/- 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 +/- 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high-affinity state (KD = 2.30 +/- 1.16 nM) and a low-affinity state (KD = 1.220 +/- 230 nM). Guanosine 5'-(3-O-thio)triphosphate or IAP treatment reduced the number of the high-affinity state binding sites without altering the KD for PIA. Apomorphine (100 microM) increased the KD value 10-fold and decreased Bmax by approximately 20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high-affinity to low-affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.     

Characterization of the Solubilized A1 Adenosine Receptor from Rat Brain Membranes

A1 adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A1 adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-N6-[3H]phenylisopropyladenosine([3H]PIA) with KD values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A1 adenosine receptors could be labelled not only with the agonist [3H]PIA but also with the antagonist 1,3-diethyl-8-[3H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein Ni and that all regulatory functions are retained on solubilization.     

Characterization of the human T lymphocyte adenosine receptor: comparison of normal and systemic lupus erythematosus cells

To determine whether a defect in the T cell response to adenosine exists at the level of the adenosine receptor in systemic lupus erythematosus (SLE) we measured the binding affinity and maximum binding of T cell membranes from both normal and SLE T cells by utilizing radiolabeled adenosine ligands. Normal T lymphocyte membranes possess a single class of [3H]5-N-ethylcarboxamide adenosine binding sites with a Kd of 0.61 microM, a Bmax of 23.5 pmol/mg protein, and a Hill coefficient of 0.98, which indicates the presence of noncooperative sites. In contrast, T cell membranes do not bind significant amounts of either [3H]cyclohexyladenosine or [3H]phenylisopropyladenosine. These data indicate that T lymphocyte membranes have only A2, and not A1, adenosine receptors. Similarly, T cells from both active and inactive SLE subjects also express only A2 receptors with a Kd of 0.93 microM, a Bmax of 20.4 pmol/mg protein, and a Hill coefficient of 0.85, which is consistent with the presence of noncooperative sites. There is no difference in the on-rate, affinity, or density of T cell A2 receptors from active SLE patients, inactive SLE patients, or healthy controls. We conclude that T lymphocytes from both healthy and SLE subjects express A2, but not A1, receptors. Thus, the inability of SLE T cells to respond to adenosine does not reflect a decreased density of A2 (stimulatory) receptors, diminished A2 receptor binding, or an increased affinity or number of A1 (inhibitory) adenosine receptors. These observations support the conclusion that the defect in the T cell cAMP-dependent pathway may occur at a point distal to the adenosine receptor.     

Discrete Distributions of Adenosine Receptors in Mammalian Retina

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N-ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that approximately 30% of the [3H]NECA binding was at non-A1,A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.     

A1 Adenosine Receptor-G Protein Coupling in Bovine Brain Membranes: Effects of Guanine Nucleotides, Salt, and Solubilization

The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAC) but not agonist (R-N6-[3H]phenylisopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Adenosine Receptor in Rat Pineal Gland: Evidence for A-2 Selectivity

We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyladenosine [( 3H]CHA), R-N6-[3H]phenylisopropyladenosine [( 3H]R-PIA), and 5'-N-ethylcarboxamido[3H]adenosine [( 3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A-1-selective ligands (CHA and R-PIA) had less than or equal to 10% specific binding. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 nM) or R-PIA (50 microM). To characterize this binding, we used the N-ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 +/- 10 nM and an apparent Bmax of 1,060 +/- 239 fmol/mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM) (-25%), a result suggesting the involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA greater than 2-chloroadenosine greater than S-adenosyl-L-homocysteine greater than CHA. Binding was also displaced by 3-isobutyl-1-methylxanthine (IC50 = 23.6 microM). These findings are consistent with the selective labeling by [3H]NECA of an A-2-type adenosine receptor in rat pineal membranes.     

Radioiodination of p-hydroxyphenylisopropyladenosine: development of a new ligand for adenosine receptors

dl-p-Hydroxyphenylisopropyladenosine has been iodinated with 125I and tested as a ligand of adenosine receptors in membranes from rat brain using a filtration assay. Binding studies using l-[3H]phenylisopropyladenosine as a ligand were carried out in parallel, and the binding of both ligands could be displaced by dl-p-hydroxyphenylisopropyladenosine and l-phenylisopropyladenosine in a similar fashion. These data establish the feasibility of using radioiodinated derivatives of adenosine as ligands of suitable types of adenosine receptors.     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

Adenosine Receptors in Myelin Fractions and Subtractions: The Effect of the Agonist (R)-Phenylisopropyladenosine on Myelin Membrane Microviscosity

In this article the existence of A1 adenosine receptors and the absence of A2 adenosine receptors in myelin membranes purified from pig brain white matter are demonstrated. The characterization of (R)-[3H]phenylisopropyladenosine ([3H]R-PIA) binding to purified myelin fractions was performed. The distribution of high- and low-affinity species of the A1 adenosine receptor was different in heavy, medium, and light myelin. The fluidity of myelin subfractions and of pig brain cortical membranes was estimated; the microviscosity of heavy myelin (5.4 poises) and of cortical membranes (5.1 poises) was similar and less than that of medium (7.8 poises) and light (8.2 poises) myelin. It was also demonstrated that the agonist R-PIA modifies the microviscosity of myelin membranes and that the degree of modification depends on the fluidity of the membrane assayed. These results suggest that adenosine receptors may have an important role in the functionality of myelin membranes.     

Nucleoside transport in heart: species differences in nitrobenzylthioinosine binding, adenosine accumulation, and drug-induced potentiation of adenosine action

The site-specific binding of the potent and selective nucleoside transport inhibitor, [3H]nitrobenzylthioinosine (NBMPR), to the nucleoside transport system of cardiac membranes of several species was investigated. The affinity of [3H]NBMPR for these sites ranged from 0.03 nM in rat to 0.78 nM in dog. The maximal binding capacity of cardiac membranes for [3H]NBMPR was also species dependent and was greatest in bovine and guinea pig heart (2551 and 1700 fmol/mg protein, respectively) and least in rat (195 fmol/mg protein). The affinities of recognized nucleoside transport inhibitors and benzodiazepines for these transport inhibitory sites in guinea pig and rat heart were estimated by studying the inhibition of the site-specific binding of [3H]NBMPR in competition experiments. These values were compared with their inhibitory effects on the transporter-dependent accumulation of [3H]adenosine in guinea pig and rat cardiac muscle segments and with their ability to potentiate the negative inotropic action of adenosine in electrically driven guinea pig and rat left atria. In guinea pig heart, the recognized nucleoside transport inhibitors and benzodiazepines had an order of affinity (dilazep greater than hydroxynitrobenzylthioguanosine greater than dipyridamole greater than hexobendine much greater than lidoflazine much greater than flunitrazepam greater than diazepam greater than lorazepam greater than flurazepam) for the NBMPR site which was similar to those for the inhibition of [3H]adenosine accumulation and for potentiation of adenosine action. In contrast, in rat heart, where the maximal binding capacity of [3H]NBMPR was lower (eightfold), the nucleoside transporter dependent accumulation of [3H]adenosine was also lower (sixfold) and the negative inotropic action of adenosine was not significantly potentiated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of adenosine binding sites in atrial sarcolemmal membranes

The studies reported here involve an exploration of the sites on atrial myocyte membranes with which adenosine interacts to produce its potent physiological effects in atrial muscle. Specific, high affinity binding of the stable adenosine analogs 2-chloro[3H]adenosine (2-ClAdo) and [3H]adenosine 5'-N-ethylcarboxamide (NECA) to atrial sarcolemmal membranes was measured in kinetic and equilibrium studies at 4 degrees C and 35 degrees C. Analysis of the [3H]2-ClAdo binding isotherm indicated the presence of two classes of binding site with equilibrium Kassoc values estimated to be 5.7 X 10(7) M-1 and 2.7 X 10(6) M-1. Displacement of bound [3H]2-ClAdo by adenosine 5'-N-cyclopropylcarboxamide (NCPCA) and by several N6-substituted adenosine analogs confirmed the presence of two classes of binding site. Analysis of the [3H]NECA binding also revealed the presence of two types of binding site for this ligand. The methylxanthines isobutylmethylxanthine and theophylline displaced bound [3H]2-ClAdo whereas adenosine uptake inhibitors and several other purines showed little activity. These atrial membrane binding sites exhibit many of the characteristics of the physiological adenosine receptors studied in intact atria. Furthermore, the [3H]2-ClAdo binding sites were sensitive to treatment with proteolytic enzymes, suggesting that these sites exist on sarcolemmal membrane proteins.     

Chronic caffeine consumption increases the number of brain adenosine receptors

Caffeine, a potent central stimulant, is known to competitively inhibit the specific binding of both adenosine and benzodiazepine receptor ligands to brain membranes in vitro. In mice receiving a diet containing non-toxic doses of caffeine (200 or 400 mg/kg diet) for periods up to 40 days, a dose-related increase in the number of binding sites for [3H]-CHA and [3H] DPX was observed in whole brain membranes without modifications of the receptors' affinity. Furthermore, a transitory increase in the number of [3H]-DZP binding sites was observed. These preliminary data seem to confirm the involvement of the adenosine receptors in the mode of action of caffeine and may be relevant to the development of both tolerance and dependence to some of the central effects of this compound.     

Characteristics of an adenosine A1 binding site in human placental membranes

Binding sites were solubilized from human placental membrane using 1.5% sodium cholate and were assayed using polyethylene glycol precipitation. These soluble binding sites had properties of an adenosine A1 binding site. 2-[3H]Chloroadenosine and N-[3H]-ethylcarboxamidoadenosine (NECA) binding were time dependent and reversible. Scatchard plots indicate two classes of binding sites with Kd values of 6 and 357 nM for 2-chloro[8-3H]adenosine and 0.1 and 26 nM with [3H]NECA. The specificity of [3H]NECA binding was assessed by the ability of adenosine analogs to complete for binding sites. Using this approach the estimated IC50 values were 60 nM for (R-PIA), 160 nM for S-PIA, 80 nM for NECA, and 20 nM for 2-chloroadenosine. Binding of [3H]NECA to the soluble sites is inhibited to 48% of the control value by 100 microM guanylyl-5'-imidodiphosphate (Gpp(NH)p). The IC50 value for NECA binding to the soluble binding site was increased from 80 nM to 1500 by Gpp(NH)p. There was a shift of binding affinity from a mixture of high and low affinity to only low affinity with 100 microM Gpp(NH)p. Despite these alterations a NECA prelabeled molecular species of 150 kDa did not decrease in molecular weight upon the addition of 100 microM Gpp(NH)p during high-performance liquid chromatography on a Superose 12 column. Other evidence to support the concept of preferential solubilization and assay of a small population of A1 binding sites was obtained. Following solubilization adenosine A2-like binding sites could be detected only in reconstituted vesicles. The existence of small amounts of A1 binding sites in intact human placental membranes was directly demonstrated using the A1 agonist ligand N6-[3H]cyclohexyladenosine and the A1 antagonist ligand 8-[3H]cyclopentyl-1,3-dipropylxanthine. JAR choriocarcinoma cells have "A2-like" membrane binding sites. In contrast to placental membranes, only A2-like binding sites could be solubilized from JAR choriocarcinoma cells. These observations indicate that human placental membranes contain adenosine A1 binding sites in addition to A2-like binding sites. These sites are guanine nucleotide sensitive, but do not shift to a lower molecular weight form upon assumption of a low affinity state.     

The Adenosine Receptors Present on the Plasma Membrane of Chromaffin Cells Are of the A2b Subtype

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.     

Influence of development and reduction of fat stores on the antilipolytic alpha 2-adrenoceptor in hamster adipocytes: comparison with adenosine and beta-adrenergic lipolytic responses

The response of the hamster adipocyte to various lipolytic (beta-adrenergic) and antilipolytic (alpha(2)-adrenergic and adenosine-dependent) stimuli was studied during the development and after cold-induced regression of fat stores. Alpha(2)-adrenergic binding ([(3)H]clonidine binding sites) was also investigated. Adipocytes came from young animals (4-5 weeks), adults (20-25 weeks), and adults submitted to a 6-week cold exposure (6 degrees C) that promoted a large decrease in fat stores and in fat cell size. The lipolytic response induced by isoproterenol (beta-agonist) was equivalent in the different groups. Adenosine and alpha(2)-adrenergic antilipolytic effects were estimated through the inhibition of theophylline-induced lipolysis by phenylisopropyladenosine and clonidine, respectively. The adenosine effect was unchanged in all the groups. In contrast, the alpha(2)-adrenergic effect, which was not present in young hamsters, increased simultaneously with fat cell size, was fully effective in adult hamsters, and had completely disappeared in small adipocytes from cold-exposed hamsters. In fat cell ghosts, alpha(2)-adrenoceptors ([(3)H]clonidine binding sites), followed similar modifications: they increased with fat cell enlargement and disappeared after cell size reduction following cold exposure. These results suggest that: 1) the increased alpha(2)-adrenergic antilipolytic response which is concomitant with fat cell enlargement could partly explain the growth-related decrease in the previously reported lipolytic effect of epinephrine; 2) the alpha(2)-receptivity of the adipocyte seems to be strictly fat cell size-dependent while the beta-adrenergic and adenosine responses are unaffected; and 3) the regulation in the adipocytes of the adenosine, alpha(2)- and beta-receptors seems to be unrelated.-Carpene, C., M. Berlan, and M. Lafontan. Influence of development and reduction of fat stores on the antilipolytic alpha(2)-adrenoceptor in hamster adipocytes: comparison with adenosine and beta-adrenergic lipolytic responses.     

Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat

Hypothyroidism is associated with an enhanced sensitivity of rat fat cells to the inhibitory action of adenosine and adenosine agonists. The sensitivity of the forskolin-stimulated cyclic AMP response of rat fat cells to the adenosine agonist N6-phenylisopropyladenosine is amplified 3-fold by hypothyroidism. Forskolin-stimulated adenylate cyclase activity is more sensitive to inhibition by this adenosine agonist in membranes of fat cells isolated from hypothyroid as compared to euthyroid rats. Hypothyroidism does not significantly alter the number of affinity of binding sites for N6-cyclohexyl[3H]adenosine or N6-phenylisopropyladenosine in membranes of rat fat cells. GTP-induced inhibition of forskolin-stimulated adenylate cyclase was markedly enhanced in the hypothyroid state, suggesting an alteration in the inhibitory regulatory component (Ni)-mediated control of adenylate cyclase. Incubating membranes with [alpha-32P]NAD+ and preactivated pertussis toxin results in the radiolabeling of two peptides with Mr = 40,000 and 41,000 as visualized in autoradiograms of polyacrylamide gels run in sodium dodecyl sulfate. The amount of label incorporated by pertussis toxin into these two peptides (putative subunits of Ni) per mg of protein of membrane is increased 2-3-fold in the hypothyroid state. The amount of the stimulatory regulatory component, Ns, in fat cell membranes is not altered by hypothyroidism (Malbon, C. C., Graziano, M. P., and Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260). The amplified response of hypothyroid rat fat cells to the inhibitory action of adenosine appears to reflect a specific increase in the activity and abundance of Ni.     

Effect of some potent adenosine uptake inhibitors on benzodiazepine binding in the CNS

A series of nucleoside transport inhibitors has been tested for their ability to displace [³H]diazepam binding to CNS membranes. No correlation between their potency as [³H]adenosine uptake blockers and as inhibitors of [³H]diazepam binding was found, either in rat or guinea-pig brain tissue. Dipyridamole, a potent adenosine transport inhibitor interacted strongly (K_i = 54 nM) with peripheral-type benzodiazepine binding sites (“acceptor sites”) and was 4–5 fold weaker in displacing [³H]methylclonazepam and [³H]Ro15-1788, ligands selective for the specific central benzodiazepine “receptor”. Unlike the benzodiazepines, dipyridamole had no anticonvulsant action against metrazole-induced convulsions in mice. Ro5-4864, a benzodiazepine which selectively interacts with the peripheral-type benzodiazepine binding site, was approximately equipotent with diazepam in inhibiting [³H]adenosine uptake in brain tissue. These results do not support the idea of a very close link between high-affinity central binding sites for clinically-active benzodiazepines and the adenosine uptake site. The possibility of a connection between benzodiazepine “acceptor” sites and the membrane nucleoside transporter is discussed.     

Sensitivity of adipocyte lipolysis to stimulatory and inhibitory agonists in hypothyroidism and starvation.

The responsiveness of lipolysis to the stimulatory agonists noradrenaline, corticotropin and glucagon and to the inhibitory agonists N6-phenylisopropyladenosine, prostaglandin E1 and nicotinic acid was investigated with rat white adipocytes incubated with a high concentration of adenosine deaminase (1 unit/ml). The cells were obtained from fed or 48 h-starved euthyroid animals or from fed or starved animals rendered hypothyroid by 4 weeks of treatment with low-iodine diet and propylthiouracil. Hypothyroidism increased sensitivity to and efficacy of all three inhibitory agonists in their opposition of noradrenaline-stimulated lipolysis. Starvation decreased sensitivity to all three inhibitory agonists when opposing basal lipolysis. Hypothyroidism decreased sensitivity to noradrenaline, glucagon and corticotropin by 37-, 4- and 4-fold respectively and decreased the maximum response to these agonists by approx. 50%, 50% and 75% respectively. Starvation reversed decreases in maximum response to these agonists in hypothyroidism. Starvation in the euthyroid state increased sensitivity to glucagon and noradrenaline, but did not alter sensitivity to corticotropin. Cells from hypothyroid rats were relatively insensitive to Bordetella pertussis toxin, which substantially increased basal lipolysis in the euthyroid state.     

Adenosine analogs inhibit fighting in isolated male mice

The potent adenosine analogs N-ethylcarboxamide adenosine (NECA) and phenylisopropyladenosine (PIA) inhibit fighting and associated agonistic behaviors in isolated male mice. These effects are reversed by methylxanthines; moderate doses of NECA which inhibit fighting have minimal effects on spontaneous locomotor activity. At very low doses, both NECA and PIA increase fighting in parallel with previously reported increases of motor activity. Brain levels of [3H]-NECA and [3H]-PIA achieved at behaviorally effective doses suggest an involvement of adenosine receptors. The biochemical mechanism of adenosine receptor action with respect to fighting is unknown, but may include neuromodulatory effects on the release of other, more classical neurotransmitters.