Transient high-affinity binding of agonists to alpha 1-adrenergic receptors of intact liver cells

At alpha 1-adrenergic receptors in isolated rat liver parenchymal cells, (-)-epinephrine is potent in eliciting a maximal increase in glycogenolysis (Kact = 24 nM). This contrasts with a 100-fold lower affinity for the agonist at alpha 1-adrenergic receptors of intact hepatocytes determined from equilibrium competition assays with the alpha 1-adrenergic antagonist [3H]prazosin. We demonstrate here that agonists bind to alpha 1-adrenergic receptors of intact liver cells initially with a markedly higher affinity than under equilibrium conditions. When incubations are performed for 15 s at 37 degrees C, the affinity is more than 100-fold higher than that obtained in equilibrium (45 min) assays (IC50 = 28 +/- 3 vs 5300 +/- 400 nM for (-)-epinephrine and 32 +/- 3 vs 6100 +/- 500 nM for (-)-norepinephrine). When incubations are performed at 4 degrees C (150 min), high-affinity binding similar to that obtained in short-term incubations can also be demonstrated. In contrast, antagonist compete with similar affinities in 15 s and 45 min assays, and their dissociation constants are not affected by changes in the incubation temperature. These results indicate that agonists bind to native alpha 1-adrenergic receptors transiently with high affinity. The conversion of receptors to a state of predominantly low affinity for agonists, which occurs rapidly and irreversibly with increasing incubation at 37 degrees C, is inhibited at low incubation temperatures. It is suggested that the high-affinity configuration of the alpha 1-adrenergic receptor for agonists observed in nonequilibrium experiments or at reduced incubation temperatures represents the physiologically relevant state of the alpha 1-adrenergic receptor.     

Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulus-aggregation coupling in platelets activated with PAF-acether

Contrary to most agonists, platelet-activating factor (PAF-acether) induces a more pronounced aggregation at 22 degrees C than at 37 degrees C. A possible explanation was sought in the mechanism that couples the PAF-acether-receptor complex with exposure and occupation of fibrinogen binding sites. Comparison of studies performed at 37 degrees C with those at 22 degrees C revealed: a faster binding of [3H]PAF-acether to its receptors; more accumulation of 32P-labelled phosphatidylinositol 4-monophosphate and a slower but more abundant formation of phosphatidic acid that lasted for 5 min; a 1.4-fold increase in phosphorylation of the Mr 47,000 protein and a 2-fold increase in phosphorylation of the myosin light chain. In contrast, less secretion occurred and less [32P]phosphatidylinositol accumulated at 22 degrees C than at 37 degrees C, and also the increase in cytosolic Ca2+ content and the formation of thromboxane B2 were considerably lower. No differences were found in [32P]phosphatidylinositol 4,5-bisphosphate formation and arachidonate metabolism. Fibrinogen binding studies revealed two types of binding at both temperatures, a high-affinity and a low-affinity binding. There were 6-fold more low-affinity binding sites at 22 degrees C than at 37 degrees C, whereas high-affinity binding did not change. These data suggest that the better aggregation found at 22 degrees C is the result of exposure of an increased number of fibrinogen binding sites. The increased protein phosphorylation and phosphatidic acid accumulation and the faster binding of PAF-acether to its receptors which accompany the better aggregation responses at 22 degrees C suggest that these processes are involved in the regulation of exposure of fibrinogen binding sites.     

Muscarinic acetylcholine receptor: thermodynamic analysis of the interaction of agonists and antagonists

The thermodynamic parameters of the interaction of agonists and antagonists with heart and brain muscarinic receptors were determined. The binding of quinuclidinyl [3H]benzilate and the inhibition of quinuclidinyl benzilate (QNB) binding by agonists and antagonists were examined at temperatures between 2 degrees C and 27 degrees C. The density of specific binding sites and the relative proportions of high- and low-affinity binding components of drugs were unaffected by the temperature changes. The binding of atropine was entropy driven in brain and heart membranes. In contrast, net values of these thermodynamic parameters for QNB binding and for the high-affinity binding component of pirenzepine to brain membranes were decreased with the enhancement of the temperature. The low-affinity binding component of the agonists carbachol, oxotremorine and pilocarpine was enthalpy driven. Their high-affinity binding component was entropy driven at 2 degrees C and became enthalpy driven when the incubation temperature was increased. The guanine nucleotide Gpp[NH]p partly prevented the temperature-dependent decrease of net entropy and enthalpy values. Considering that the net changes of thermodynamic parameters are relevant of the interactions between the ligand, the receptor protein and the adjoining membranous molecules, a three-state conformational model is proposed for the muscarinic receptor protein. The receptor selectivity is reappreciated owing to these three states of the receptor protein and the different components of the muscarinic receptor complexes.     

Direct assessment of beta-adrenergic receptors in intact rat adipocytes by binding of [3H]CGP 12177. Evidence for agonist high-affinity binding complex and for beta 1 and beta 2 receptor subtypes

The characteristics of the binding of the hydrophilic beta-adrenergic antagonist [3H]CGP 12177 to intact rat adipocytes were studied at 37 degrees C and 6 degrees C. At both temperatures and at 90% saturation, the non-specific binding was less than 30% of the total binding. At 37 degrees C, specific [3H]CGP 12177 binding was rapid, reversible of high affinity (1.8 +/- 0.4 nM) and saturable. The number of specific binding sites per adipocyte increased with the fat cell size (about 35 000 and 115 000 sites per cell in adipocytes with diameters of 60 microns and 88 microns, respectively) but remained constant when expressed per unit fat cell surface area. Displacement of [3H]CGP 12177 bound to adipocytes by unselective and selective beta-antagonists was stereospecific, had the same characteristics as those found in adipocyte membranes and showed a heterogeneous specificity for beta 1 and beta 2 adrenergic subtypes. In contrast, beta agonist competition curves, which modeled to two affinity-states of binding, showed high-affinity-state Kd values for agonists 10-25-times higher than those found in membranes under the same experimental conditions. At 6 degrees C, although the number and affinity of the specific binding sites for [3H]CGP 12177 were the same as those found at 37 degrees C, the Kd value for (-)-isoproterenol binding to the high affinity state of these sites (3.0 +/- 0.5 nM) was 25-times lower than at 37 degrees C and similar to the value found in membrane preparations (1.5-4 nM). These results show that the [3H]CGP 12177 specific binding sites detected on intact adipocytes represent the physiological beta-adrenergic receptors. Moreover, this study extends to the adipocyte the validity of the model recently proposed for other cell lines, according to which in intact cells, but not in membranes, agonist-binding promotes a rapid and temperature-dependent conformational change of the beta-adrenergic receptors, leading to a progressive loss of capacity of agonists to form a high-affinity complex.     

Evidence for multiple rat VPAC1 receptor states with different affinities for agonists.

We compare the binding properties of [125I-VIP] and [125I]-Ro 25 1553 to VPAC1 receptors, expressed in stably transfected CHO cells. [125I]-VIP labelled two VPAC1 receptor states, while [125I]-Ro 25 1553 labelled selectively a limited number of high-affinity receptors. This high-affinity state probably corresponds to an agonist-receptor-Gs ternary complex as its properties (guanyl nucleotides, EC50 values and maximal effect) were affected by cholera toxin pre-treatment. Both high- and low-affinity receptors participated in the adenylate cyclase activation. This suggested that agonists activate not only low-affinity uncoupled receptors by facilitating the ternary complex formation, but also activated the high-affinity ternary complex by accelerating the GTP binding to emptied, receptor-bound G proteins.     

Characterization of [3H]Clonidine Binding to Two Sites in Calf Brain Membranes

Kinetic studies showed that under appropriate conditions, [3H]clonidine binds to two distinct receptor sites in calf cortex membranes. At 23 degrees C, binding was obtained at a low-affinity site (dissociation constant, KD = 5.4 nM) and a high-affinity site (KD = 1.1 nM). In contrast, at 0 degree C, selective binding occurred to the low-affinity site only. Consequently, at 0 degree C, it was possible to evaluate the interaction of drugs with the low-affinity receptor directly. On the other hand, competition with the high-affinity receptor could be ascertained by generating displacement curves representing the differential between specific binding values obtained at 23 and 0 degree C. Guanine nucleotides selectively decreased binding to the high-affinity site without apparent influence on the low-affinity [3H]clonidine binding. The activities of various pharmacological agents at the low- and high-affinity clonidine receptors are discussed and compared with WB-4101 binding data.     

The identification and characteristics of subpopulations of alpha 1-adrenergic receptors

Rat liver and brain alpha 1-adrenergic receptors were purified 500 fold by successive chromatographic steps using heparin- and wheat germ agglutinin-agarose; an affinity matrix constructed by coupling CP85.224 (a derivative of prazosin) to affigel 102. It is shown that the existence in brain of an alpha 1-adrenergic receptor subpopulation, which is structurally distinct from that previously characterized. Chlorethylclonidine, irreversibly inactivates [3H] prazosin binding sites in partially purified membrane preparations of rat liver. Under identical conditions, only 50% of receptors are irreversibly inactivated. Computer modelling of data obtained from the competition by the alpha-antagonists, WB 4101 and phentolamine, for [3H] prazosin binding to partially purified preparations of rat liver is best fit by assuming a single low-affinity site for both ligands. However, the partially purified brain preparations indicates the presence of two affinity binding sites for these antagonists. Prior alkylation of brain receptors with chlorethylclonydyne results in the loss of the low-affinity phentolamine and WB4101 binding sites. These data provide evidence for the existence of a single receptor subpopulation (alpha 1b) in rat liver and for two subpopulations (alpha 1a and alpha 1b) in rat brain. The significance of these results in understanding the signal mechanisms which allow cellular responsiveness to alpha 1-adrenergic receptor agonists is discussed.     

Complete Conversion of Brain D2 Dopamine Receptors from the High- to the Low-Affinity State for Dopamine Agonists, Using Sodium Ions and Guanine Nucleotide

Since previous work had shown that brain D2 3,4-dihydroxyphenylethylamine (dopamine) receptors were only partly converted from their high-affinity state to their low-affinity state, we here tested whether it was possible to obtain a complete 100% conversion of these receptors into their low-affinity state. It was first essential to resolve the components of [3H]spiperone binding to dopaminergic sites and nondopaminergic sites in rat striatal homogenates. In the presence of 50 microM S-sulpiride (to occlude the dopaminergic sites), therefore, we first determined that the residual binding of [3H]spiperone (approximately 20%) was inhibited by serotonergic agonists much more effectively than dopamine or noradrenaline, thus identifying the serotonergic component of [3H]spiperone binding. Thus, dopamine (or ADTN) inhibited the binding of [3H]spiperone at a high-affinity site (with dissociation constant of 10 nM dopamine), at a low-affinity site (with dissociation constant of 2,000 nM dopamine), and at the serotonergic site (with dissociation constant of 50,000 nM dopamine). In the absence of sodium ions, the high-affinity site was about 50% occupied by [3H]spiperone, and guanine nucleotide had no effect on this proportion. In the presence of 120 mM NaCl, however, the high-affinity site was reduced to 15% and guanine nucleotide completely eliminated this high-affinity site, 100% of the sites having been completely converted to their low-affinity state. Using [3H]N-propyl-norapomorphine to label the high-affinity state of the dopamine receptor, 50% conversion into the low-affinity state occurred at 45 mM LiCl, 69 mM NaCl, and 202 mM KCl. We conclude that it is possible to convert brain D2 dopamine receptors completely into their low-affinity state, in the presence of NaCl and a guanine nucleotide, providing that appropriate allowance is made for the serotonergic component of [3H]spiperone binding.     

Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes

Oxytocin initiates its insulin-like action in adipocytes through oxytocin-specific receptors. We have studied binding and structural properties of these receptors with the radioligand [3H]oxytocin. Steady-state binding was reached after 45 min, at 21 degrees C, and 10 min at 37 degrees C. Scatchard analyses of equilibrium binding data indicated a single class of oxytocin binding sites at 21 degrees C (KD = 3.3 nM, RT = 6 X 10(4) sites/cell) and 2 binding sites at 37 degrees C (KD = 1.5 nM, RT = 6 X 10(4) sites/cell; and KD = 20 nM, RT = 30 X 10(4) sites/cell). Insulin, insulin-like growth factor I, and epidermal growth factor increased oxytocin binding (approximately 20-40%), whereas adenosine, a regulator of oxytocin action, did not affect oxytocin binding. Binding activity of oxytocin was impaired by pretreatment of the hormone or adipocytes with dithiothreitol. Dithiothreitol treatment of adipocytes preferentially inactivated high-affinity binding sites. N-ethyl maleimide inhibited oxytocin binding in adipocytes more than dithiothreitol. In contrast to the inhibitory effects of dithiothreitol and N-ethyl maleimide, proteases (trypsin, chymotrypsin and papain) were not able to inhibit fat cell binding activity. These results suggested that in isolated adipocytes: there are high-affinity and low-affinity receptors, but the low-affinity receptors are absent at 21 degrees C; the binding of oxytocin can be regulated by insulin, and growth factors; and the oxytocin receptors contain disulfide bridges and free thiols that are essential for the maintenance of oxytocin binding.     

Specific vasopressin binding to rat adrenal glomerulosa cells. Relationship to inositol lipid breakdown.

Cells from the zona glomerulosa of rat adrenals were isolated and maintained for 3 days in primary culture. Specific vasopressin binding was determined by using [3H]vasopressin. [3H]Vasopressin binding was time-dependent (half-time of about 2 min for 6 nM free ligand) and reversible on addition of unlabelled vasopressin (80% dissociation within 30 min). Dose-dependent [3H]vasopressin binding at equilibrium indicated that vasopressin interacted with two populations of sites: high-affinity sites (dissociation constant, Kd = 1.8 nM; maximal binding capacity = 10 fmol/10(6) cells) and low-affinity sites. Vasopressin increased the cellular content of labelled inositol mono-, bis- and tris-phosphate in cells prelabelled with myo-[3H]inositol. The vasopressin concentration eliciting half-maximal inositol phosphate accumulation was very close to the Kd value for vasopressin binding to high-affinity sites. Competition experiments using agonists and antagonists with enhanced selectivity for previously characterized vasopressin receptors indicated that vasopressin receptors from rat glomerulosa cells are V1 receptors of the vascular or hepatic subtype. The detected specific vasopressin-binding sites might represent the specific receptors mediating the mitogenic and steroidogenic effects of vasopressin on glomerulosa cells from rat adrenals.     

Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low-Km GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with less than 10% of the sites in the high-affinity state. Both sites were also detected at 4 degrees C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the Bmax for [3H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

A common binding site for primary prostanoids in vascular smooth muscles: a definitive discrimination of the binding for thromboxane A2/prostaglandin H2 receptor agonist from its antagonist

Differences in binding characteristics between agonists and antagonists for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were examined in rat cultured vascular smooth muscle cells (VSMC). Scatchard analysis indicated the existence of two binding sites for the TXA2/PGH2 agonist, whereas a single class of recognition sites for the receptor antagonists were observed with approximately the same maximum binding capacity (Bmax) as a high-affinity binding site of the agonist. Weak binding inhibition by approx. 100 nM of primary prostanoids (PGE1, PGF2 alpha and PGD2) was detected only with the TXA2/PGH2 agonist, and not with the antagonist. Primary prostanoids as well as TXA2/PGH2 agonists (U46619 and STA2) suppressed the [3H]PGF2 alpha and [3H]PGE1 binding with almost the same potency, whereas TXA2/PGH2 antagonists (S-145, SQ29,548 and ONO3708) did not. The Bmax value of the binding sites was roughly identical in PGF2 alpha, PGE1 and a low-affinity binding site of U46619. These results suggest the existence of two binding sites for TXA2/PGH2 in VSMC, i.e., a high-affinity binding site corresponding to that of the TXA2/PGH2 antagonists and a low-affinity binding site in common with primary prostanoids.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.     

Electroconvulsive Shock Increases α1b-but Not α1a-Adrenoceptor Binding Sites in Rat Cerebral Cortex

Repeated administration of electroconvulsive shock (ECS) increases [3H]prazosin binding to alpha 1-adrenoceptors in rat cerebral cortex. In contrast, [3H]WB4101 binding in cortex has been reported to be unchanged after ECS. [3H]Prazosin labels two alpha 1-adrenoceptor subtypes, termed alpha 1a and alpha 1b, whereas [3H]WB4101 labels the alpha 1a subtype preferentially. The purpose of this study was to determine whether ECS increases one or both alpha 1-adrenoceptor subtypes in rat cerebral cortex. We found that treatment of rats with ECS once daily for 10-12 days increased [3H]prazosin binding in cortex by about 25% but did not significantly alter [3H]WB4101 binding to alpha 1-adrenoceptors. Measurement of alpha 1a and alpha 1b receptors by competition analysis of the selective alpha 1a antagonist 5-methylurapidil against [3H]prazosin and measurement of [3H]prazosin binding in homogenates preincubated with chlorethylclonidine, which alkylates alpha 1b binding sites, also indicated that the ECS-induced increase in alpha 1-adrenoceptors is confined to the alpha 1b subtype. In contrast to its effect on [3H]prazosin binding, ECS did not increase phosphoinositide hydrolysis as measured by [3H]inositol 1-phosphate accumulation in slices of rat cerebral cortex stimulated by either norepinephrine or phenylephrine. The failure of ECS to increase [3H]inositol 1-phosphate accumulation stimulated by phenylephrine, which is a partial agonist for this response, suggests that spare receptors do not account for the apparent absence of effect of ECS on alpha 1-adrenoceptor-mediated phosphoinositide hydrolysis.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.     

Evidence that High- and Low-Affinity DL-α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid (AMPA) Binding Sites Reflect Membrane-Dependent States of a Single Receptor

Binding of DL-alpha-[3H]amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([3H]AMPA) to lysed rat brain membranes in the presence of potassium thiocyanate resulted in curvilinear Scatchard plots that could be resolved by regression analysis into a large low-affinity component and a small high-affinity component. Solubilization with Triton X-100 resulted in solubilized and nonsolubilized fractions that were considerably enriched in the high-affinity component and correspondingly reduced in the low-affinity component. It thus appears that solubilization converts low-affinity AMPA receptors into high-affinity receptors. Also, synaptic plasma membranes were found to be greatly enriched in the low-affinity form and deficient in the high-affinity form of the AMPA receptor. These experiments provide evidence for the hypothesis that the high- and low-affinity components of AMPA binding are interconvertible states of the same receptor rather than separate binding sites and that the conversion of these receptors from their native high-affinity state to the low-affinity state occurs on insertion of the receptors into synapses.     

Studies on the hepatic alpha 1-adrenergic receptor. Modulation of guanine nucleotide effects by calcium, temperature, and age

The effects of guanine nucleotides on the hepatic alpha 1-adrenergic receptor were studied using norepinephrine (NE) displacement of [3H]prazosin binding to rat liver plasma membranes. Nonhydrolyzable GTP analogues caused large rightward shifts of norepinephrine displacement curves of [3H]prazosin binding in EGTA-treated membranes, but only small shifts in membranes prepared with Ca2+. The effect of a brief Ca2+ exposure on NE displacement curves was not reversed by adding excess EGTA prior to binding experiments. Analysis of the curves showed that the EGTA membranes had an increased number of high affinity agonist sites (Kd, 42 nM) and that guanyl-5'-yl imidodiphosphate (GppNHp) converted these to low affinity sites (Kd, 1039 nM). When binding was carried out at 2 degrees C, the norepinephrine displacement curves were shifted to the left, and GppNHp was without effect. Neither EGTA, Ca2+, nor 2 degrees C treatment altered [3H]prazosin binding per se. Attempts were made to differentiate the potency order of GTP analogues which alter glucagon receptor binding (presumably mediated by the stimulatory GTP-binding protein, Na, of the adenylate cyclase system) from the potency order of GTP analogues which alter alpha 1-receptor agonist binding (presumably mediated by a yet uncharacterized GTP-binding protein which some have speculated may be distinct from Ns). However, the potency series of GTP analogues to alter norepinephrine binding was GTP gamma S greater than GppNHp greater than or equal to GTP greater than or equal to GDP greater than or equal to GppCHp greater than GMP (where GTP gamma S represents guanosine 5'-O-(thiotriphosphate) and GppCHp represents guanyl-5'-yl (beta, gamma-methylene)diphosphonate) and was identical to that for inhibition of [125I]iodoglucagon binding. The ability of GppNHp to alter norepinephrine displacement of [3H]prazosin binding increased with the age of the rat from which membranes were prepared. This was due to the fact that juvenile rats (50-75 g) had few alpha 1-receptors in the high affinity state, whereas in old rats (430-490 g) more of the receptors were in this form. Age has previously been shown to increase alpha 1-adrenergic stimulation of cAMP in isolated hepatocytes (Morgan, N.G., Blackmore, P. F., and Exton, J. H. (1983) J. Biol. Chem. 258, 5103-5109) but did not affect the dose-response curves for norepinephrine-induced Ca2+ mobilization and phosphorylase activation in these cells. These data suggest that alpha 1-adrenergic receptors can become coupled to a guanine nucleotide-responsive moiety in hepatic plasma membranes and that this may be similar to Ns.(ABSTRACT TRUNCATED AT 400 WORDS)     

On high- and low-affinity agonist sites in GABAA receptors

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.