Modulation of GABA transport by adenosine A1R-A2AR heteromers, which are coupled to both Gs- and G(i/o)-proteins.

Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A(1) (A(1)R) and A(2A) (A(2A)R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A(1)R-A(2A)R that signal via two different G-proteins, G(s) and G(i/o), and either enhances (A(2A)R) or inhibits (A(1)R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane.

A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins.

ZM 241385, an adenosine A(2A) receptor antagonist, inhibits hippocampal A(1) receptor responses.

4-(2-[7-amino-2-(2-furyl¿1,2,4¿-triazolo¿2,3a¿-¿1,3, 5¿triazin-5-yl-amino]ethyl)phenol (ZM 241385) has been used as an antagonist of adenosine A(2A) receptors, exhibiting high selectivity over adenosine A(1) receptors. We now report that ZM 241385 (10-50 nM) attenuated the inhibitory action of N(6)-cyclopentyladenosine (10 nM) and R(-)-N(6)-phenylisopropyladenosine (R-PIA, 20 nM), two selective adenosine A(1) receptor agonists, on hippocampal population spike amplitude. This effect is unlikely to be a direct antagonism of adenosine A(1) receptor since the K(i) of ZM 241385 to displace [3H]PIA (2 nM) binding, from hippocampal membranes ranged from 0.8 to 1.9 microM. These results question the usefulness of ZM 241385 to define adenosine A(2A) receptors actions in functional studies.

Comparative pharmacology of human adenosine receptor subtypes - characterization of stably transfected receptors in CHO cells.

Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands.

Diimidazo[1,2-c:4',5'-e]pyrimidines: adenosine agonist activity demonstrated by microphysiometry.

Silicon-based microphysiometry, measuring extracellular acidification rate of cells in culture, demonstrated that a series of diimidazo[1,2-c:4',5'-e]pyrimidines were agonists at the human adenosine A1 receptor. 5-amino-7,8-dihydro-3-ribofuranose-8-(R)-(phenyl)-3H-diimidazo [1,2-c:4',5'-e]pyrimidine (2a) had an EC50 of 100 microM and reached 90% of the Emax produced by R-PIA.

Diimidazo[1,2-c:4',5'-e]pyrimidines: N6-N1 conformationally restricted adenosines.

Tethering the N6-substituents of N6-substituted adenosines to N1 has resulted in a series of conformationally restricted adenosine analogues. The resultant diimidazo[1,2-c:4',5'-e]pyrimidines were shown to be adenosine A1 selective.

Short term desensitization of the A1 adenosine receptors in DDT1MF-2 cells.

Previous studies have indicated that desensitization of the A1 adenosine receptor (A1AR), unlike other adenosine receptor subtypes and G protein-coupled receptors, required prolonged exposure to agonists. We more closely studied this observation by focusing on changes in the A1AR signal transduction pathway after short term agonist exposure (0.5-4 hr) in the hamster vas deferens smooth muscle cell line (DDT1MF-2 cells). Incubation of these cells with 1 microM (R)-phenylisopropyladenosine [(R)-PIA] produced a time-dependent loss in binding of the agonist radioligand [125I]N6-2-(4-amino-3-iodophenyl)ethyladenosine but not of the antagonist radioligand [3H]8-cyclopentyl-1,3-dipropylxanthine. This was accompanied by a reduction in the high affinity (G protein-coupled) state of this receptor from 63 +/- 8% to 37 +/- 12% after treatment for 4 hr. Moreover, cells treated with (R)-PIA demonstrated reduced agonist-stimulated GTPase activity and diminished inhibition of adenylyl cyclase activity but no change in expression of alphai and beta subunits. The decreases in agonist binding in the desensitized cells were reversible after treatment of DDT1MF-2 cell membranes with alkaline phosphatase or protein phosphatases 1 and 2A, suggesting a role of phosphorylation in the uncoupling and desensitization of the A1AR. Incubation of cells with (R)-PIA led to rapid translocation of G protein-coupled receptor kinase (GRK) from the cytosol to the plasma membrane within 1 hr of exposure. In addition, purified preparations of the A1AR that were phosphorylated with purified recombinant GRK-2 demonstrated enhanced affinity for arrestin over Gi/Go. These results indicate rapid and functional desensitization of the A1AR by brief exposure to agonist. The mechanism underlying this event seems to involve phosphorylation of the A1AR, presumably by the GRK or GRKs.

Expression and function of adenosine receptors in the chinchilla cochlea.

Previous studies indicate the presence of adenosine receptors in the cochlear tissues obtained from different animals. This study was initiated to determine the subtypes of adenosine receptor (AR) present in the chinchilla cochlea and to assess their function. Radioligand binding studies demonstrate the presence of both the A1AR and A3AR in membranes prepared from the cochlea, using the radioligands [3H]DPCPX and [125I]APNEA. Estimates of the number (Bmax) of A1AR and A1AR plus A3AR by saturation curves were 118 +/- 13 and 417 +/- 120 fmol/mg, respectively, with the respective equilibrium dissociation constants (Kd) averaging 2.7 +/- 0.2 and 26.3 +/- 13.8 nM. No significant number of A2aAR were detected using [3H]CGS21680. The nonhydrolyzable adenosine analog R-phenylisopropyladenosine (R-PIA, 1 microM) elicited a small but significant degree of inhibition of forskolin-stimulated adenylyl cyclase activity (10.4 +/- 2.5%) in cochlear membrane preparations, which was insensitive to blockade by theophylline (100 microM). Furthermore, R-PIA elicited an increase in inositol 1,4,5-trisphosphate production in dissociated cell preparations obtained from the cochlea. No significant effect of R-PIA was observed on auditory measures such as auditory brainstem evoked response, cochlear action potential and endocochlear potential following round window application. However, round window application of R-PIA elicited significant increases in the activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase and significantly reduced the levels of malondialdehyde, a marker of lipid peroxidation. These results suggest a potential cytoprotective role of adenosine in the cochlea against oxidative damage.

[3H]-(R)-N6-Phenylisopropyladenosine agonist binding to the solubilized A1 adenosine receptor from bovine epididymal spermatozoa.

The high-affinity agonist radioligand [3H]-(R)-N6-phenylisopropyladenosine ([3H]-R-PIA) was used to investigate agonist A1 adenosine receptors interactions in soluble preparations from bovine spermatozoa. The digitonin-solubilized receptor shows a high-affinity state with a Kd of 5.32+/-1.17 nM and a Bmax of 460+/-33 fmol/mg protein. The binding capacity, higher than that of the membrane-bound form, indicates that the soluble preparation is likely enriched with binding sites. In the presence of guanylyl-5'-imidodiphosphate (Gpp(NH)p), [3H]-R-PIA binds to the soluble receptor with a Kd of 7.97+/-1.44 nM and a Bmax of 400.8+/-27 fmol/mg protein. The radioligand rapidly dissociates with a K(-1) of 0.125 min(-1) although specific [3H]-R-PIA is still found in solubilized A1 receptor. The A1 agonist N6-cyclopentyladenosine differentiates two affinity states, whereas Gpp(NH)p shifts the agonist curve to the right and all the receptors are in the low-affinity state. In the presence of NaCl, the agonist still recognizes two affinity states with a lower affinity than that observed in the absence of NaCl. Analyses of sedimentation profiles show the existence of a population of A1 receptors tightly coupled to Gi, the pertussin toxin-sensitive component of the G protein family.

Reconstitution of bovine A1 adenosine receptors and G proteins in phospholipid vesicles: betagamma-subunit composition influences guanine nucleotide exchange and agonist binding.

We have studied the interactions of purified A1 adenosine receptors and G proteins reconstituted into phospholipid vesicles to investigate how the betagamma composition of G protein heterotrimers influences coupling. Recombinant hexahistidine-tagged bovine A1 adenosine receptors were expressed in Sf9 cells and purified to homogeneity by sequential chromatography over heparin-sepharose, xanthine amino congener-agarose, and nickel-nitrilotriacetic acid columns. These receptors were reconstituted with pure recombinant G proteins of defined subunit composition. Receptor-G protein complexes containing alphai2 and beta1gamma2 or beta1gamma3 and stimulated with the agonist, (R)-phenylisopropyladenosine, exchange guanine nucleotide 2-3 times more rapidly than do complexes containing beta1gamma1. This difference is not overcome by increasing the concentration of betagamma subunits. Receptor-G protein complexes containing beta1gamma1 also bind less of the agonist, [125I]-iodoaminobenzyladenosine (125I-ABA), than do complexes containing beta1gamma3. Kinetic experiments show that 125I-ABA dissociates 2-fold more rapidly from receptor-G protein complexes containing beta1gamma1 than from complexes containing the other betagamma subunits. The affinity of the interaction between immobilized Galphai2 subunits and beta1gamma1 or beta1gamma2 measured with an optical biosensor in the absence of receptor is similar. Taken together, these data implicate the gamma-subunit in influencing the interaction between the A1 adenosine receptor and G proteins.

Modulation of GH4 cell cycle via A1 adenosine receptors.

Identification and characterization of A1 adenosine receptors (A1Rs) in a tumor cell line derived from rat pituitary (GH4 cells) was performed by ligand binding and immunocytochemistry. Subsequently, the involvement of A1Rs in the regulation of cell proliferation was studied in these cells. The agonist N6-(R)-phenylisopropyladenosine (R-PIA) did not modify the number of cultured cells, but it regulated the kinetics of the cell cycle. By means of experiments of pulse and of pulse and chase with bromodeoxyuridine and further labeling with Hoechst 33258, propidium iodide, and/or fluorescein-conjugated antibodies against bromodeoxyuridine, it was demonstrated that R-PIA, via A1Rs, accelerated progression from G0/G1 to S phase and from S to G2/M phase of the cell cycle, whereas the initiation of a new cycle occurred at the same time in treated and untreated cells. As a consequence, R-PIA did not change the total length of the cycle. This is the first description of cell cycle regulation without modification of cell proliferation. Although pertussis toxin blocked the R-PIA-induced inhibition of cyclic AMP production in these cells, it did not affect the R-PIA action on the cell cycle. In contrast, cholera toxin mimicked the action of R-PIA. Thus, it is likely that regulation of the cell cycle via A1Rs is mediated by heterotrimeric G proteins different from those that mediate inhibition of adenylate cyclase. Due to the fact that cells in G0/G1 phase were less susceptible to secretory signals, adenosine, in an autocrine manner and by regulating the cell cycle kinetics, may contribute to the modulation of the secretory capacity of pituitary cells.

Untreated Graves' but not remission Graves' immunoglobulin G preparations increase intracellular Ca2+ in FRTL-5 thyroid cells.

We compared immunoglobulin G (IgG) preparations obtained from untreated Graves' patients with those from Graves' patients in remission and normal subjects in terms of their activity to stimulate the phospholipase C-Ca2+ signaling. Ca2+ mobilizing activity of the untreated Graves' IgG preparations in FRTL-5 thyroid cells was statistically (P < 0.01) and significantly higher than the activity of normal IgG preparations, whereas there was no significant difference in the activity between the remission Graves' and normal IgG preparations. Digital video imaging of fura2-loaded FRTL-5 cells confirmed that the Ca2+ mobilizing action of the untreated Graves' IgG preparations is an intracellular event. Phospholipase C activation by the untreated but not remission Graves' IgG preparations was statistically higher than that by normal IgG preparations. Involvement of the phospholipase C activation in the Ca2+ response mechanism was confirmed by the enhancement of the Ca2+ response by an adenosine derivative, N6(L-2-phenylisopropyl)adenosine (PIA) which can potentiate agonist-induced phospholipase C activation but not the Ca2+ mobilization itself. The Ca2+ response to the IgG preparations did not show a significant correlation with their cAMP response (TSAb). Therefore, the Ca2+ response to Graves' IgG preparations may be utilized as a functional marker for Graves' disease in addition to TSAb.

Synthesis and structure-activity relationship of pyrazolo[3,4-d]pyrimidines: potent and selective adenosine A1 receptor antagonists.

A series of 12 substituted 1-phenylpyrazolo[3,4-d]pyrimidines were synthesized and evaluated for rat brain adenosine A1 and A2a receptor binding affinity. Substituents at C-4 and C-6 were varied in order to define these regions in terms of molecular recognition by the receptor subtypes. At C-4, the effects of a mercapto, methylthio, and amino substituent were evaluated, while at C-6, amides with varying alkyl groups extending from the alpha-carbon were examined. This study identified both potent and selective adenosine A1 receptor antagonists. The most potent of the 12 compounds was alpha-[(4-amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl)thio]hexanamide (14); with an A1 Ki of 0.939 nM and an A2a Ki of 88.3 nM, this compound is 94-fold A1 selective. The most selective of the 12 compounds was alpha-[[4-(methylthio)-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl]thio]hex anamide (10); with an A1 Ki of 6.81 nM and an A2a Ki > 40 000 nM, this compound is > 5900-fold A1 selective. The structure-activity relationships for the complete series has identified discrete structural differences between the A1 and A2a receptors with respect to the binding of pyrazolo[3,4-d]pyrimidines. This study resulted in prediction that increased A1 affinity could be achieved by incorporation of NH-alkyl substituents at C-4. This was confirmed by synthesis of alpha-[[4-(methylamino)-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl]thiol] hexanamide (15) which was found to have an A1 Ki of 0.745 nM.

Inhibition of receptor/G protein coupling by suramin analogues.

Suramin analogues act as direct antagonists of heterotrimeric G proteins because they block the rate-limiting step of G protein activation (i.e., the dissociation of GDP prebound to the G protein alpha subunit). We have used the human brain A1 adenosine receptor and the rat striatal D2 dopamine receptor, two prototypical Gi/G(o)-coupled receptors, as a model system to test whether the following analogues suppress the receptor-dependent activation of G proteins: 8-(3-nitrobenzamido)-1,3,5-naphthalenetrisulfonic acid (NF007), 8-(3-(3-nitrobenzamido)-benzamido)-1,3,5-naphthalenetrisulfonic acid (NF018); 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalenetr isulfonic acid) (NF023); 8,8'-(carbonylbis(imino-3,1-phenylene)carbonylimino-(3,1- phenylene)) bis(1,3,5-naphthalenetrisulfonic acid) (NF037); and suramin. Suramin and its analogues inhibit the formation of the agonist-specific ternary complex (agonist/receptor/G protein). This inhibition is (i) quasicompetitive with respect to agonist binding in that it can be overcome by increasing receptor occupancy but (ii) does not result from an interaction of the analogues with the ligand binding pocket of the receptors because the binding of antagonists or of agonists in the absence of functional receptor/G protein interaction is not affected. In addition to suppressing the spontaneous release of GDP from defined G protein alpha subunits, suramin and its analogues reduce receptor-catalyzed guanine nucleotide exchange. The site, to which suramin analogues bind, overlaps with the docking site for the receptor on the G protein alpha subunit. The structure-activity relationships for inhibition of agonist binding to the A1 adenosine receptor (suramin > NF037 > NF023) and of agonist binding to the inhibition D2 dopamine receptor (suramin = NF037 > NF023 > NF018) differ. Thus, NF037 discriminates between the ternary complexes formed by the agonist-liganded D2 dopamine receptors and those formed by the A1 adenosine receptor with > 10-fold selectivity. Therefore, our results also show that inhibitors can be identified that selectively uncouple specific receptor/G protein tandems.

The cluster-arranged cooperative model: a model that accounts for the kinetics of binding to A1 adenosine receptors.

To explain the equilibrium binding and binding kinetics of ligands to membrane receptors, a number of models have been proposed, none of which is able to adequately describe the experimental findings, in particular the apparent negative cooperativity of ligand binding. In this paper, a new model, the cluster-arranged cooperative model, is presented whose main characteristic is that it explains the existence of negative cooperativity in the binding of ligands to the receptor molecule. The model is based on our findings of agonist binding to A1 adenosine receptors and of ligand-induced clustering of these receptors on the cell surface. The model assumes the existence of two conformational forms of the receptor in an equilibrium which depends on the concentration of the ligand. In this way, negative cooperativity is explained by the transmission of the information between receptor molecules through the structure of the membrane. The model is able to predict the thermodynamic binding and binding kinetics of [3H]-(R)-(phenylisopropyl)adenosine to A1 adenosine receptors in the presence and absence of guanylyl imidodiphosphate. In the presence of the guanine nucleotide analogue, the linear Scatchard plots obtained for [3H]-(R)-(phenylisopropyl)adenosine binding are explained by the disappearance of cooperativity, thus suggesting that G proteins are important for the existence of negative cooperativity in ligand binding. Among other predictions, the model justifies early events in homologous desensitization since high ligand concentrations would lead to the saturation of the receptor in a low-affinity conformation that does not signal. Our model can likely explain the behavior of a number of heptaspanning and tyrosine-kinase receptors exhibiting complex binding kinetics.

Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors.

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A1 adenosine receptor (A1R) is a second ecto-ADA binding protein. Binding of ADA to A1R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A1R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via A1R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via A1R.

Evidence of A1 adenosine receptor on epidydimal bovine spermatozoa.

3H-R-phenylisopropyladenosine (PIA) was used to characterize adenosine receptors on bovine epidydimal spermatozoa membranes. Dypiridamole, an adenosine uptake inhibitor, did not effect the radioligand binding, indicating an external site for the interaction of adenosine with spermatozoa. Steady-state binding was achieved after 45 min at 25 degrees C and lasted for at least 3 h. Scatchard plots were linear with a Kd of 6.98 +/- 1.02 nM and Bmax of 34 +/- 8 fmol/mg protein. N-6-cyclopentyladenosine (CPA), with a Ki of approx. 0.196 nM, was the most potent inhibitor of binding and the agonist order potency series was CPA > R-PIA = N-6-cyclohexyladenosine > N-6-phenyladenosine > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > 2-(p-2-carboxyethyl)phenylamine)-5'-N-ethylcarboxy-amidoa denosine. 1-3-Dipropyl-8-cyclopentylxanthine (DPCPX), an A1 receptor selective antagonist, produced the strongest inhibition with a Ki of 0.46 +/- 0.1 nM. Antagonist order potency series DPCPX > xanthine amine congener > cyclopentyltheophilline = theophylline > caffeine > 1-3-dipropylxanthine > 8-phenyltheophilline was consistent with A1 adenosine receptor (A1AR). Guanylyl-5'-imidodiphosphate did not decrease bound 3-H-R-PIA nor accelerate its dissociation, a behavior consistent with inhibitory receptors only. The incubation of isolated membranes with N-ethylmaleimid followed by a reduction of 57% of the ligand binding further supports the existence of A1AR on bovine epidydimal spermatozoa.

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes.

The binding characteristics of radiolabeled N6-(cyclohexyl)adenosine ([3H]CHA), N6-(R-phenylisopropyl)adenosine ([3H]R-PIA), 5'-N-ethyl-carboxamidoadenosine ([3H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5'-N-ethylcarboxamidoadenosine ([3H]CGS 21680), to rat testis membranes were investigated. Specific binding of [3H]CGS 21680, a selective agonist for the A2a adenosine receptor, was very modest whilst the nonselective agonist [3H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [3H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the A1 adenosine receptor, [3H]CHA and [3H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A1 adenosine receptor with a potency order for agonists of CHA > or = R-PIA > NECA > N6-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A3 adenosine receptor in these membranes we selectively blocked the A1 receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [3H]CHA and [3H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA > or = NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A3 adenosine receptor.

Effect of somatotropin treatment on lipogenesis, lipolysis, and related cellular mechanisms in adipose tissue of lactating cows.

The effect of bST on the metabolism of lipid in adipose tissue was studied using tissue biopsies from lactating cows treated with bST for 8 d. Cows responded to treatment by increasing daily milk yield by 10.9 kg, although net energy intake was not changed. Thus, net energy balance was changed from highly positive to slightly negative (+7.7 to -1.1 Mcal/d). Consistent with these changes in net energy balance, lipogenesis rates were dramatically reduced (97%) in adipose tissue from bST-treated cows. Activities of acetyl-coenzyme A carboxylase (initial and total) and fatty acid synthase were also dramatically decreased. Therefore, for cows in positive energy balance, reduced lipid synthesis in adipose tissue represents a major mechanism whereby bST alters nutrient partitioning to support greater milk synthesis. Treatment with bST had no effect on beta-adrenergic-stimulated lipolysis in adipose tissue explants incubated in vitro with adenosine deaminase. However, bST treatment reduced the ability of adenosine to inhibit lipolysis in adipose tissue, which involved changes in both sensitivity and responsiveness to adenosine. Therefore, the enhanced lipolytic response to catecholamine in vivo with bST treatment relates to relief in the adenosine inhibitory signaling cascade rather than to a direct effect on the stimulatory signaling pathway.

Ascorbate/Fe(3+)-induced peroxidation and inhibition of the binding of A1 adenosine receptor ligands in rat brain membranes.

The effect of peroxidation induced by the ascorbate/Fe3+ system on the binding properties of the A1 adenosine receptor, was studied in rat brain membranes, using the agonist, [3H]R-N6-phenylisopropyladenosine ([3H]R-PIA), and the antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX). For the agonist, as well as for the antagonist, the number of binding sites (Bmax) was significantly (P < 0.05) reduced after pretreatment of the membranes with ascorbate/Fe3+. The affinity of the agonist for the binding sites was not statistically modified (P > 0.05) after ascorbate/Fe3+ pretreatment, whereas the Kd value of the antagonist was increased (P < 0.05) by a factor of 2. Ascorbate/Fe3+ pretreatment affected agonist binding in the presence of GTP in a similar way as that observed in the absence of GTP, suggesting that peroxidation also affects agonist binding to A1 adenosine receptors uncoupled to G-proteins. The results suggest that when brain membranes suffer free radical oxidative damage, the adenosine modulation of neuronal activity through A1 receptors could be less efficient.