Effect of serum, alpha-1 acid glycoprotein, lipoproteins and albumin on human mononuclear leucocyte beta-adrenoceptors

The effects of serum, alpha-1 acid glycoprotein (AAG), serum lipoproteins (SLP) and human serum albumin (HSA) on 3H-(-)-dihydroalprenolol (3H-(-)-DHA) binding and (-)-isoproterenol ((-)-IPR) induced cyclic AMP (cAMP) elevation in human peripheral blood mononuclear leucocytes (MNL) were investigated. The saturable binding of 3H-(-)-DHA was decomposed into two classes of binding sites with maximum binding capacity of approximately 1400 and 30000 sites/cell and with dissociation constants (Kd) of approximately 0.7 and 65 nM. Stimulation of the MNL beta-adrenoceptors by (-)-IPR caused a concentration dependent cAMP accumulation (EC50 approximately 0.2 microM) with maximum level approximately 250% above basal. For all single leucocyte preparations, 30-35 min. exposure to serum, AAG and SLP increased the number of beta-adrenoceptors with 100-200% and the maximal responsiveness to (-)-IPR with 30-90%. The presence of proteins did not change the Kd or the EC50. (-)-Alprenolol inhibited concentration dependently the serum induced increment in (-)-IPR-responsiveness. Serum, AAG and SLP did also increase the number of low affinity binding sites with 25-40% without effect on the Kd. HSA had no consistent effect on beta-adrenergic binding or stimulation. The present study shows that serum, AAG and SLP influence the number and function of MNL beta-adrenoceptors in vitro.     

Binding of prazosin to alpha 1-acid glycoprotein and albumin: effect of protein purity and concentrations

Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93-95% was observed at 37 degrees for therapeutic drug concentrations. Both alpha 1-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd approximately 0.8 microM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd approximately 30 microM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd approximately 8 microM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.     

Fluorescence studies of beta-adrenergic ligand binding to alpha 1-acid glycoprotein with 1-anilino-8-naphthalene sulfonate, isoprenaline, adrenaline and propranolol.

The present study shows that ANS (1-anilino-8-naphthalene sulfonate), propranolol, isoprenaline, adrenaline and dopamine have common binding sites on AAG (alpha 1-acid glycoprotein). A fluorescence technique was employed to characterize the interaction between the ligands and AAG at 20-22 degrees. The binding of ANS to AAG caused increased fluorescence intensity at emission and excitation wavelengths of 400 and 470 nm. In this situation, propranolol displaced ANS in a concentration-dependent mode with an apparent dissociation constant of 6.2 +/- 0.01 microM, whereas isoprenaline did not reduce the ANS-AAG fluorescence. However, in the presence of AAG, catecholamines caused a marked increase of fluorescence at excitation and emission wavelengths of 250 and 325 nm, respectively. These wavelengths were employed to characterize the binding of isoprenaline, adrenaline and propranolol to AAG. Two subsets of binding sites were demonstrated. The Kd values were 0.87 +/- 0.03 and 25.1 +/- 10.7 microM for ANS, 0.76 +/- 0.09 and 133 +/- 30.4 microM for propranolol, 140 +/- 14 and 2.18 +/- 0.58 mM for isoprenaline, 137 +/- 24 and 14.8 +/- 0.1 mM for adrenaline, respectively. AAG had identical high affinity binding capacity for these ligands (n approximately 1). However, the second class of binding sites showed ligand-dependent binding capacity: n = 1 for ANS, n approximately 10 for propranolol, n approximately 15 for adrenaline, n approximately 20 for isoprenaline, respectively. ANS, propranolol, dopamine and adrenaline caused concentration-dependent inhibition of isoprenaline binding to AAG with apparent dissociation constants of 5.1 +/- 1.8 microM, 6.4 +/- 1.1 microM, 0.57 +/- 0.13 mM and 1.5 +/- 0.46 mM, respectively.     

Binding of Prazosin to α1-Acid Glycoprotein and Albumin: Effect of Protein Purity and Concentrations

Abstract: Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93–95% was observed at 37° for therapeutic drug concentrations. Both α₁-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd≈0.8 μM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd≈30 μM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd≈8 μM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.     

Effect of Serum, α-1 Acid Glycoprotein,Lipoproteins and Albumin on Human Mononuclear Leucocyte β-Adrenoceptors

Abstract: The effects of serum, α-1 acid glycoprotein (AAG), serum lipoproteins (SLP) and human serum albumin (HSA) on ³H-(-)-dihydroalprenolol (³H-(-)-DHA) binding and (-)-isoproterenol ((-)-IPR) induced cyclic AMP (cAMP) elevation in human peripheral blood mononuclear leucocytes (MNL) were investigated. The saturable binding of ³H-(-)-DHA was decomposed into two classes of binding sites with maximum binding capacity of approximately 1400 and 30000 sites/cell and with dissociation constants (K_d) of approximately 0.7 and 65 nM. Stimulation of the MNL β-adrenoceptors by (-)-IPR caused a concentration dependent cAMP accumulation (EC50 ～0.2 μM) with maximum level approximately 250% above basal. For all single leucocyte preparations, 30–35 min. exposure to serum, AAG and SLP increased the number of β-adrenoceptors with 100–200% and the maximal responsiveness to (-)-IPR with 30–90%. The presence of proteins did not change the K_d or the EC50. (-)-Alprenolol inhibited concentration dependently the serum induced increment in (-)-IPR-responsiveness. Serum, AAG and SLP did also increase the number of low affinity binding sites with 25–40% without effect on the K_d. HSA had no consistent effect on β-adrenergic binding or stimulation. The present study shows that serum, AAG and SLP influence the number and function of MNL β-adrenoceptors in vitro.     

The effect of the plasticizers TBEP (tris-(2-butoxyethyl)-phosphate) and DEHP (di-(2-ethylhexyl)-phthalate) on beta-adrenergic ligand binding to alpha 1-acid glycoprotein and mononuclear leukocytes

The plasticizers tris-(2-butoxyethyl)-phosphate (TBEP) and di-(2-ethylhexyl)-phthalate (DEHP) and the beta-adrenergic receptor-blockers [3H]-(-)-dihydroalprenolol ([3H]-(-)-DHA) and [3H]-(-)-CGP 12177 were tested for their ability to interact with beta-adrenergic binding to alpha 1-acid glycoprotein (AAG) and mononuclear leukocytes (MNL). The IC50-values, obtained by displacement of [3H]-(-)-DHA bound to AAG, were 3.5 nM, 2 microM and 4 microM for TBEP, (-)-alprenolol and DEHP, respectively. (+/-)-CGP 12177 had virtually no effect on radioligand binding to AAG. The [3H]-(-)-CGP 12177 binding to MNL consisted of beta-adrenergic receptor binding (Kd = 210 pM) and non-saturable binding. [3H]-(-)-DHA was bound to two different classes of binding sites on MNL, the beta-adrenergic receptors (Kd = 440 pM) and a secondary class of binding sites (Kd = 64 nM). (+/-)-CGP 12177 displaced about 30% of [3H]-(-)-DHA from MNL with an IC50-value of 190 pm. (-)-ALP displaced about 85% of total bound radioligand and gave a biphasic displacement curve with IC50-values of 320 pM and 690 mM, respectively. TBEP displaced a considerable fraction of [3H]-(-)-CGP 12177 and [3H]-(-)-DHA bound to MNL beta-adrenergic receptors, whereas DEHP had no effect. In contrast, DEHP caused displacement of [3H]-(-)-DHA from the MNL low affinity sites, but was a markedly less potent displacer compared to TBEP. The present study shows that TBEP and DEHP interact with beta-adrenergic transport proteins, non-specific tissue binding sites and beta-adrenergic receptors coupled to adenylate cyclase. Plasticizers may thus affect the biology and pharmacology of the beta-adrenergic signal system.     

Tianeptine binding to human plasma proteins and plasma from patients with hepatic cirrhosis or renal failure.

1. The binding of tianeptine to human plasma, isolated plasma proteins, red blood cells and to plasma from patients with cirrhosis or renal failure was studied in vitro by equilibrium dialysis. 2. Tianeptine is highly bound to plasma (95%) at therapeutic concentrations (0.3-1 microM). No saturation of the binding sites was seen. 3. Human serum albumin (HSA) was shown to be mainly responsible for this binding (94%) with a saturable process characterized by one binding site with a moderate affinity (Ka = 4.2 x 10(4) M-1) and a non-saturable process with a low total affinity (nKa = 1.2 x 10(4) M-1). 4. Like many basic and amphoteric drugs, tianeptine showed a saturable binding to alpha 1-acid glycoprotein (AAG) with one site and a moderate affinity (Ka = 3.7 x 10(4) M-1). Its binding to lipoproteins and red blood cells (RBC) was weak and non-saturable. Over the range of therapeutic drug concentrations (0.3-1 microM), the unbound fraction in blood remains constant (4.5%). 5. Interactions were studied using non-esterified fatty acids (NEFA) at pathological concentrations; they altered tianeptine binding to plasma and to isolated HSA. Tianeptine seems to bind to a HSA site different from sites I (warfarin) and II (diazepam), but close to site II. It also shares the only basic-site on AAG. However, at therapeutic drug concentrations (0.3-1 microM), not all of these interactions occur. 6. The binding of tianeptine varied according to HSA, AAG and NEFA concentrations both in patients and healthy subjects. In patients with chronic renal failure having high NEFA concentrations the unbound fraction of tianeptine (fu) increased from 0.045 to 0.153 compared with normal (P less than 0.001). In cirrhotic patients, with relatively low HSA concentrations, the fu of tianeptine increased from 0.045 to 0.088 compared with normal (P less than 0.01). 7. Multiple regression analysis of all of the data indicated that the fu of tianeptine was related significantly to HSA, NEFA and AAG concentrations, with a particularly strong correlation with NEFA concentrations. Therefore, variation of HSA and NEFA concentrations in patients on maintenance therapy may cause an increase of tianeptine fu.     

Guanine nucleotides regulate both agonist and antagonist binding to cod brain alpha 1-adrenoceptors

The effect of guanine nucleotides on the binding of 3H-prazosin and its displacement by various agonists and antagonists were studied in membranes prepared from the cod brain. When cod brain membranes were maintained in a buffer containing 8 mM Mg2+, GTP (1 mM) was found to increase the specific binding of 3H-prazosin Computer modelling 3H-prazosin saturation curves, suggested that the number of binding sites for 3H-prazosin was increased by 18 +/- 5% by GTP without affecting the affinity for 3H-prazosin (dissociation constant, Kd, 56 +/- 3 pM). Displacement of 3H-prazosin by adrenaline produced shallow displacement curves. Computer modelling these curves suggested that adrenaline bound to two sites - one high and one low affinity site - the Kd's being 0.14 +/- 0.03 (KH) and 7.6 +/- 0.5 microM (KL), respectively. When 1 mM GTP was present the displacement curves were shifted to the right and became steeper. Computer modelling these curves suggested that adrenaline now bound to only one low affinity site with a Kd of 7.6 +/- 0.5 microM. When unlabelled prazosin was used to displace 3H-prazosin the displacement curves were uniphasic and steep, irrespective of whether GTP was present or not. Computer modelling these curves suggested that prazosin bound to only one site with a Kd of 68 +/- 11 pM. In the absence of Mg2+ and in the presence of EDTA (0.8 mM) the displacement curve of adrenaline became steeper and the effect of GTP was almost abolished. During these conditions the ability of GTP to enhance 3H-prazosin binding was also abolished.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of 1-anilino-8-naphthalene sulfonate as a fluorescent probe in the investigation of drug interactions with human alpha-1-acid glycoprotein and serum albumin

We report a rapid method for the characterization of the human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) interactions with drugs. The binding of 1-anilino-8-naphthalene sulfonate (ANS) to AAG and HSA was measured by fluorescence spectroscopy. Fluorescence data indicated that ANS was bound tightly to at least one site on AAG, with an affinity constant of 1.35 x 10(6) M-1. The fluorescence of an ANS:AAG complex was quenched by the binding of various drugs. Fluorescence quenching of the HSA:ANS complex showed a single site with an affinity constant of 0.72 x 10(6) M-1. The interaction of AAG and HSA with ANS or other drugs was also studied by comparative equilibrium dialysis. [14C]Pipequaline was used as an AAG and HSA site marker. [14C]Pipequaline seems to share sites I (azapropazone) and II (diazepam and ibuprofen) of HSA. However, high concentrations of warfarin were unable to displace [14C]pipequaline. On the other hand, it was shown that palmitic acid decreased, whereas bilirubin increased the pipequaline binding.     

Serum binding of indapamide in health and disease: primary role of alpha 1-acid glycoprotein

The serum concentrations of alpha-1-acid glycoprotein (AAG), albumin (HSA), and non-esterified fatty acids (NEFA), and the serum binding of indapamide were measured in four groups of individuals: control (healthy) subjects (N = 24), patients with inflammatory syndrome (N = 28), with hepatic (N = 20) and renal (N = 27) insufficiency. Indapamide serum binding was increased in patients with inflammatory syndrome (82.2 +/- 3.4%, P less than .001), decreased in patients with hepatic insufficiency (72.3 +/- 5.9%, P less than .001) and unchanged in patients with renal insufficiency (77.7 +/- 2.8%) as compared with controls (78.2 +/- 3.1%). A multivariate analysis indicated that these changes were mainly related to concomitant changes in AAG concentration (that explained 63% of intersubject variability in bound/free binding ratio), and to a lesser extent to HSA (that explained only 4% of the variability in the binding). These data show that the free fraction of the acidic drug indapamide in serum is affected by pathologic conditions in which changes in AAG concentration occur and that, unexpectedly, HSA plays a negligible role in the binding.     

Binding of cosalane--a novel highly lipophilic anti-HIV agent--to albumin and glycoprotein

Cosalane is a potent inhibitor of HIV replication with multiple sites of action. The purposes of this study were to (a) determine the extent and nature of cosalane binding to mucin, alpha(1)-acid glycoprotein (AAG), plasma, and human (HSA) and bovine serum (BSA) albumin, and (b) determine the primary site(s) of cosalane binding to HSA. Plasma protein binding of cosalane was studied by a gel filtration technique. Cosalane binding to HSA was also determined in the presence of salicylic acid. Competitive inhibition studies were conducted using warfarin, digitoxin, and diazepam to determine the primary HSA binding site(s) of cosalane. The drug was bound extensively to HSA and BSA and required 500-550 moles to saturate 1 mole of protein. Stoichiometries of cosalane binding to alpha(1)-acid glycoprotein (AAG) and mucin were between 30 and 50 mol/mol of either glycoprotein. The binding isotherm deviated from a rectangular hyperbola, suggesting self-association of the ligand. Salicylic acid decreased cosalane binding to HSA by one order of magnitude. Inhibition studies of cosalane to HSA revealed that the compound binds primarily to warfarin site with a K(i) of 1.24 +/- 0.24 nM. In summary, cosalane binds extensively to serum albumins and to a lesser extent to both AAG and mucin.     

High affinity binding of 3H rauwolscine and 3H RX781094 to alpha 2 adrenergic receptors and non-stereoselective sites in human and rabbit brain cortex membranes

The radiolabeled antagonists 3H RX 781094 and 3H rauwolscine bind with high affinity to alpha 2 adrenergic receptors as well as to non-receptor sites in human and rabbit brain cortex membranes. These non-receptor sites form an important contaminant of the specific binding when non-specific binding is determined in the presence of 10 microM phentolamine or more. While phentolamine is no suitable ligand to discriminate both sites, (-)-epinephrine displays a sufficient affinity ratio to separate radioligand binding to these sites. When 1 microM (-)-epinephrine is used for the determination of the non-specific binding, both radioligands bind specifically to alpha 2 receptors. Under these conditions, 3H rauwolscine and 3H RX 781094 bind to the same amount of non-cooperative sites; binding isotherms for human brain are Bmax = 113 +/- 15 fmol/mg protein and Kd = 22.8 +/- 4.2 nM for 3H RX781094 and Bmax = 110 +/- 17 fmol/mg protein and Kd = 4.7 +/- 2.5 nM for 3H rauwolscine. Competition binding experiments show, for both radioligands and in both species, the typical pharmacological potency order of alpha 2 adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin for the antagonists and UK 14304 greater than p-aminoclonidine greater than or equal to (-)-epinephrine greater than (+)-epinephrine greater than isoproterenol for the agonists. Whereas the alpha 2 receptor sites display high affinity and stereoselectivity towards (-)-epinephrine and (+)-epinephrine, the non-receptor sites bind both epinephrine isomers with equal low affinity. Specific binding of both radioligands to these sites can be determined when total binding is performed in the presence of 1 microM (-)-epinephrine and non-specific binding the presence of 1 mM phentolamine. 3H rauwolscine binding to the non-stereoselective sites can be displaced with high affinity by 5-HT, suggesting binding to a 5-HT1-receptor. The 3H RX 781094 binding displays low affinity for most alpha adrenergic ligands and do not correspond to beta adrenergic, dopaminergic or serotonergic receptors.     

Characterization of alpha 2-adrenergic receptors of calf retina membranes by [3H]-rauwolscine and [3H]-RX 781094 binding

Alpha 2-adrenergic receptors were identified in calf retina membranes by binding of the radiolabelled antagonists [3H]-RX 781094 and [3H]-rauwolscine. When 10 microM phentolamine was used to determine the non-specific binding, both radioligands labelled a single class of non-cooperative sites: Bmax = 1051 +/- 252 fmol/mg protein, Kd = 5.1 +/- 1.5 nM for [3H]-RX 78104 and Bmax = 1167 +/- 449 fmol/mg protein, Kd = 21.0 +/- 4.1 nM for [3H]-rauwolscine. Competition binding experiments showed the typical pharmacological potency order of alpha 2-adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin. Agonist competition binding curves revealed the presence of two receptor populations, having respectively high affinity (70% of the total receptor population) and low affinity for agonists, but with the same affinity for the antagonists. The high affinity sites could be converted into low affinity sites by guanine nucleotides. The non-specific binding of [3H]-RX 781094 was the same if 0.1 mM (-)-epinephrine was used instead of phentolamine. In contrast, the non-specific binding of [3H]-rauwolscine was markedly lower with (-)-epinephrine than with phentolamine. Under this condition, the Scatchard plot of [3H]-rauwolscine saturation binding was curvilinear, indicating the presence of low affinity sites for the radioligand in addition to alpha 2-adrenergic receptors. Competition binding experiments revealed that these low affinity sites were distinct from adrenergic receptors: the catecholamine agonists (-)- and (-)-epinephrine, (-)-norepinephrine, (-)-isoproterenol and dopamine competed with similar Ki values (microM range) whereas clonidine did not interact. Furthermore, these sites bound reserpine and the alpha 2-adrenergic antagonists yohimbine and rauwolscine but not phentolamine.     

Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site.     

Protein binding of nifedipine

The protein binding of nifedipine in concentrations up to 1200 ng ml-1 has been measured in serum, pure human albumin solution and pure human alpha 1-acid glycoprotein (AAG) solutions by ultrafiltration. The drug was extensively bound in serum from four healthy volunteers with a mean (+/- s.d.) fraction bound of 0.992 +/- 0.008. In albumin solution (40 g litre-1) the mean (+/- s.d.) fraction bound 0.970 +/- 0.012, was not significantly different (P greater than 0.05) from that in serum, suggesting that albumin is the major, but not necessarily the only, binding protein for nifedipine in serum. The binding of nifedipine in solutions of AAG was proportional to the AAG concentration and ranged from 0.514 +/- 0.059 to 0.755 +/- 0.035 in solutions containing 50 and 150 mg % AAG, respectively. Binding of nifedipine in all protein solutions was linear.     

Alpha 2-adrenoceptor subtypes and imidazoline-like binding sites in the rat brain.

1. The binding of [3H]-yohimbine and [3H]-idazoxan to rat cortex and hippocampus is rapid, reversible and of high affinity. Saturation data indicate that a single population of binding sites exist for [3H]-yohimbine in the cortex (Bmax 121 +/- 10 fmol mg-1, protein; Kd 5.2 +/- 0.9 nM) and hippocampus (Bmax 72 +/- 6 fmol mg-1 protein; Kd 5.8 +/- 0.7 nM). [3H]-idazoxan labels one site in the cortex (Bmax 87 +/- 8 fmol mg-1 protein; Kd 4.1 +/- 0.9 nM) and hippocampus (Bmax 30 +/- 6 fmol mg-1 protein; Kd 3.5 +/- 0.5 nM), when 3 microM phentolamine is used to define non-specific binding. A second distinct [3H]-idazoxan binding site (Bmax 110 +/- 21 fmol mg-1 protein; Kd 3.6 +/- 0.07 nM) is identified in rat cortex if 0.3 microM cirazoline is used to define non-specific binding and 3 microM yohimbine is included to prevent binding to alpha 2-adrenoceptors. 2. Displacement studies indicate that the alpha 1-adrenoceptor antagonist prazosin and the 5-HT1 ligands 8-OH-DPAT, RU 24969 and methysergide differentiate [3H]-yohimbine binding into two components; a high and low affinity site. In contrast the displacement of [3H]-idazoxan by each ligand was monophasic. 3. The affinities of 8-OH-DPAT, RU 24969 and methysergide determined against [3H]-idazoxan binding to the cortex and hippocampus correlate significantly with the binding site displaying low affinity for prazosin and previously designated alpha 2A. In contrast, a poor correlation exists for the high affinity site for prazosin designated alpha 2B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential binding of disopyramide and warfarin enantiomers to human alpha(1)-acid glycoprotein variants

AIMS: The F1S and A genetic variants of alpha1-acid glycoprotein (AAG) change under various physiological and pathological conditions. They also vary in their drug binding abilities. We have studied the stereoselective binding ability of each of the AAG variants using enantiomers of disopyramide (DP) and warfarin (WR). METHODS: The AAG variants were separated by hydroxyapatite chromatography. Binding of drug enantiomers to the AAG variants was studied by the Hummel-Dreyer method. The characteristics of the binding activities were examined by Scatchard plot analysis. The first five amino-terminal amino acids (residues 112-116) of the cyanogen bromide (CNBr) fragment (residues 112-181) of each of the separated AAG fractions were elucidated by Edman degradation. RESULTS: Commercial AAG was separated into two main fractions. Residues 112-116 of fraction 2 were identical to the amino acid sequences predicted from the AAG A gene, LAFDV, and encode the F1S variant. In fraction 3, the deduced amino acid sequence of the AAG B gene, FGSYL, was established, and encodes the A variant. The binding affinities of both DP enantiomers in fraction 3 were significantly higher than those in fraction 2. The differences between dissociation constants (Kd) in fractions 2 and 3 were 5.2-fold for (S)-DP (P < 0.05) and 3.7-fold for (R)-DP (P < 0.001). The dissociation constant of (S)-DP (0.39 +/- 0.08 micro m) was lower than that of (R)-DP (0.53 +/- 0.10 micro m) in fraction 3 [95% confidence interval (CI) - 0.282, - 0.010; P < 0.05], although the binding activities of the DP enantiomers were almost the same in fraction 2. By contrast WR enantiomers had a higher binding affinity in fraction 2 than in fraction 3, the differences in dissociation constants between fractions 2 and 3 being 12.6-fold for (S)-WR (P < 0.001) and 8.3-fold for (R)-WR (P < 0.001). The dissociation constant of (S)-WR (0.28 +/- 0.10 microm) was significantly lower than that of (R)-WR (0.48 +/- 0.08 microm) in fraction 2 (95% CI - 0.369, - 0.028; P < 0.05), but there were no significant differences between the binding activities of WR enantiomers in fraction 3. CONCLUSIONS: DP and WR enantiomers bind preferentially to fraction 3 and fraction 2, respectively. Fractions 2 and 3 are encoded by the AAG A and the AAG B genes, respectively.     

Adenosine receptors in basolateral membranes of rat renal cortex.

High affinity binding sites for adenosine were identified in rat kidney cortex basolateral membranes. Kinetic analysis indicates two sets of [3H]adenosine, [3H]ADO, binding sites, one with high affinity and Kd = 0.84 +/- 0.25 microM, one with low affinity and Kd = 4.74 +/- 0.37 microM. The ADO receptors were further characterized using ADO analogs as binding inhibitors. The most potent inhibitor of [3H]ADO binding was N-methyl-adenosine with a Kd of 5 microM, whereas 2-deoxyadenosine was about 50 times less potent. The binding of [3H]phenylisopropyladenosine, [3H]PIA, and [3H]-N-ethylcarboxamidoadenosine, [3H]NECA, to basolateral membranes was rapid and reversible. The Scatchard plot of [3H]PIA binding showed monophasic curves for experiments performed at 0 degrees C and 37 degrees C. The apparent Kd of [3H]PIA binding at 0 degrees C was 0.19 +/- 0.05 nM and 0.34 +/- 0.07 nM at 37 degrees C. The binding of [3H]NECA to basolateral membranes was found with an apparent affinity Kd of 110 +/- 50 nM at 0 degrees C. Pretreatment of membranes with N-ethylmaleimide (NEM) inhibited the [3H]PIA binding and did not affect the [3H]NECA binding. These results demonstrate that both A1 and A2 adenosine receptors are present in basolatertal membranes of rat kidney.     

Plasma protein binding of quinine: binding to human serum albumin, alpha 1-acid glycoprotein and plasma from patients with malaria.

The binding of quinine to human serum albumin (HSA), alpha 1-acid glycoprotein (AAG) and plasma obtained from healthy subjects (10 caucasians and 15 Thais) and from Thai patients with falciparum malaria (n = 20) has been investigated. In healthy volunteers, plasma protein binding expressed as the percentage of unbound quinine was 7.9-31.0% (69-92.1% bound). The mean percentage of unbound quinine found with essentially fatty acid-free HSA (40 g L-1) was 65.4 +/- 1.5% (mean +/- s.d.) and was comparable with the value (66.3 +/- 3.8%, mean +/- s.d.) for Fraction V HSA (40 g L-1). This suggests that fatty acids do not influence the plasma protein binding of quinine. Binding of quinine to 0.7 g L-1 AAG was high (mean unbound 61.0 +/- 5.0%), indicating that quinine is bound primarily to AAG and albumin, although other plasma proteins such as lipoproteins may be involved. The mean percentage of unbound quinine was slightly less in caucasians (14.8 +/- 6.7% unbound), compared with healthy Thai subjects (17.0 +/- 6.7% unbound). The higher binding of quinine in caucasian subjects was associated with a higher plasma AAG concentration observed in caucasians. Mean percentage of unbound quinine was significantly lower in Thai patients with malaria (10.9 +/- 4.0%) than in the healthy Thai subjects. The increase in the extent of quinine binding corresponded with the increase in the acute-phase reactant protein, AAG in the patients with malaria. Overall, when the data were combined there was a significant correlation (r = 0.846, P < 0.005) between the binding ratio (bound/unbound) of quinine and the plasma AAG concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5.

Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)