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It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine [(R)-AHPIA] into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels [Mol. Pharmacol. 30:403-409 (1986)]. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling, which is described in the accompanying paper [Mol. Pharmacol. 39:524-530 (1991)].

This study describes experiments investigating the mechanism of activation of A1 adenosine receptors. Isolated rat fat cells were used as a cellular model. The A1 receptors of these cells were covalently labeled with the agonist photoaffinity label R-2-azido-N6-p-hydroxyphenylisopropyladenosine. The covalent incorporation of the label into the binding subunit of the receptor was verified by demonstration of specific labeling of a peptide with Mr = 35,000 by the radioiodinated label. Such covalent labeling followed by removal of label not covalently bound led to a concentration-dependent reduction of cellular cAMP levels. This persistent effect of covalent labeling occurred with an IC50 value of 9 nM compared to an IC50 value of 0.9 nM for the direct reduction of cAMP levels by the label. The affinity of the label was determined in binding experiments. The Ki value of 19 nM was about 20 times higher than the corresponding IC50 value of cAMP reduction. Finally, the comparison between covalent binding and its effects suggests that covalently labeled receptors were fully activated. The data are interpreted as evidence for a receptor activation according to the occupancy theory. The analysis of the various concentration-response curves reveals the presence of spare receptors, which can be demonstrated by the method of agonist photoaffinity labeling.

A\(_1\) 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 A\(_1\) adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-\(N^6\)-[\(^3\)H]phenylisopropyladenosine([\(^3\)H]PJA) with K\(_D\) 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 A\(_1\) adenosine receptors could be labelled not only with the agonist [\(^3\)H]PIA but also with the antagonist I ,3-diethyi-8-[\(^3\)H]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 N; and that all regulatory functions are retained on solubilization. Key Words: A1 adenosine receptors - Solubilization- Rat brain membranes. Klotz K.-N. et al. Characterization of the solubilized A1 adenosine receptor from rat brain membranes. J. Neurochem. 46, 1528-1534 (1986).

Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors.

The binding of agonists and antagonists to Ri adenosine receptors of synaptosomal membranes from rat and bovine brain was studied. The effects of guanine nucleotides and temperature were analyzed with the aid of computerized curve fitting. Evidence is presented for two different states of the receptor: one of high and one of low affinity for agonists. Antagonists bind to both states with the same affinity. The two states are characterized by saturation, competition, and kinetic experiments with very similar results. Guanine nucleotides cause transition of the high- to the low-affinity state. The ratio of the KD values for the two affinity states is 90-150 in rat brain but only 10 in bovine brain. The proportions of the two affinity states are the same for all agonists tested; in the absence of exogenous guanine nucleotides, 75% of the total receptor population is in the high-affinity state, whereas in the presence of guanine nucleotides only 5% remain in the high-affinity state. Binding of antagonists to the receptor is enthalpy-driven whereas binding of the agonist (-)-N6-phenylisopropyladenosine to the high-affinity state of the receptor is entropy-driven. Binding of the agonist to the low-affinity state is enthalpy-driven and thus similar to the binding of antagonists. Our data indicate that guanine nucleotides convert the Ri adenosine receptor from a high- to a low-agonist affinity state and that agonist binding shows thermodynamic differences from antagonist binding only when it is to the high-affinity state of the receptor.

The binding of agonists and antagonists to alpha 2-adrenergic receptors of human platelets was studied. The receptors showed homogeneous affinities for antagonists but two affinity states for the agonist (-)-epinephrine, which were modulated by guanine nucleotides. Van't Hoff plots of antagonist binding had a break point at about 18 degrees and considerable diversity between 18 degrees and 0 degree. Agonist binding to both affinity states showed a similar break point; agonist binding to the high affinity state was characterized by a large entropy component compared to the low affinity state. This entropy component was reduced at higher concentrations of sodium, indicating that it may be due to liberation of sodium ions. Measurements of the fluorescence of 1-anilin-8-naphthalenesulfonate showed thermotropic phase transitions of the platelet membranes at about 17 degrees. The transition temperature was decreased to about 12 degrees by addition of 10 mM octanoic acid. Octanoic acid also shifted the break points of the van't Hoff plot of antagonist and low affinity agonist binding from 18 degrees to 12 degrees. High affinity agonist binding, however, remained unchanged. It is concluded that agonist-specific thermodynamic characteristics of ligand binding to alpha 2-receptors of human platelets can only be investigated by regarding differences between high and low affinity agonist binding. These differences include an entropy increase upon ligand binding, which is in part due to enhanced liberation of sodium ions, and a loss of sensitivity to fluidity changes in the outer layer of the plasma membrane.

No abstract available.

The binding of agonists and antagonists to a2-adrenergic receptors of human platelets was studied. The receptors showed homogeneaus affinities for antagonists but two affinity states for the agonist (-)-epinephrine, which were modulated by guanine nucleotides. Van't Hoffplots of antagonist binding had a break point at about 18° and considerable diversity between 18° and 0°. Agonist binding to both affinity states showed a similar break point; agonist binding to the high affinity state was characterized by a large entropy component compared to the low affinity state. This entropy component was reduced at higher concentrations of sodium, indicating that it may be due to Iiberation of sodium ions. Measurements of the fluorescence of 1-anilin-8-naphthalenesulfonate showed thermotropic phase transitions of theplatelet membranes at about 17°. The transition temperature was decreased to about 12° by addition of 1 0 mM octanoic acid. Octanoic acidalso shifted the break points of the van't Hoffplot of antagonist and low affinity agonist binding from 18° to 12°. High affinity agonist binding, however, remained unchanged. It is concluded that agonist-specific thennodynamic characteristics of ligand binding to a2-receptors of human platelets can only be investigated by regarding differences between high and low affinity agonist binding. These differences include an entropy increase upon Iigand binding, which is in part due to enhanced liberation of sodium ions, and a loss of sensitivity to fluidity changes in the outer layer of the plasma membrane.

The positive inotropic effect of the alpha-1 adrenoceptor agonist phenylephrine was accompanied by a concentration-dependent increase in inositol trisphosphate (IP3) in electrically driven left auricles isolated from rat hearts. Further analysis of the myocardial phosphoinositide pathway revealed an additional increase in inositol phosphate and inositol bisphosphate with a concomitant decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. The decrease in phosphatidylinositol bisphosphate and increase in IP3 preceded the increase in force of contraction. All effects were antagonized by the alpha-1 adrenoceptor antagonist prazosin. For comparison the effects of the beta adrenoceptor agonist isoprenaline were studied. Isoprenaline produced a positive inotropic effect similar to that of phenylephrine but all phosphoinositide products remained unaffected. The influence of lithium and calcium ions were studied systematically. The stimulatory effect of phenylephrine on inositol phosphates was lithium-dependent. Without lithium phenylephrine did not detectably affect the phosphoinositide turnover. Phenylephrine caused a maximal increase in inositol phosphate, inositol bisphospate and IP3 at 10 mmol/l of lithium. Lithium itself had a concentration-dependent positive inotropic effect. However, lithium did not enhance the positive inotropic effect of phenylephrine. Variation of the extracellular concentration of calcium did not influence the stimulatory effect of phenylephrine on inositol phosphates indicating that inositol phosphate turnover does not require the presence of extracellular calcium. It is concluded that the stimulation of myocardial phosphoinositide breakdown generating an increased IP3 turnover may be involved in the mechanism(s) whereby alpha-1 adrenoceptor stimulation exerts an increase in myocardial force of contraction.