Binding of the Adenosine A2 Receptor Ligand [3H]CGS 21680 to Human and Rat Brain: Evidence for Multiple Affinity Sites

A new radiolabeled adenosine receptor agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadeno sin e (CGS 21680), apparently specific for high-affinity binding sites of the A2 subtype in rat brain, was used to identify and pharmacologically characterize adenosine receptors in human brain. The binding of [3H]CGS 21680, as determined by standard radioligand binding technique in the presence of exogenously added adenosine deaminase, reached equilibrium after 40 min at 25 degrees C. In saturation studies, a single class of high-affinity binding sites with values for KD of 22 +/- 0.5 nM and Bmax of 444 +/- 63 fmol/mg of protein were observed. Similar binding characteristics were observed regardless of whether rapid filtration or centrifugation was used to separate bound versus free ligand. Of the 14 brain regions examined, [3H]CGS 21680 binding was highest in putamen, followed by globus pallidus and caudate nucleus. The level of [3H]CGS 21680 binding in these areas of basal ganglia was identical to 5'-N-[3H]ethylcarboxamidoadenosine ([3H]NECA) binding in the presence of 50 nM N6-cyclopentyladenosine (CPA). The rank order of agonist potencies as determined by a series of competition experiments was NECA greater than or equal to CGS 21680 greater than 2-chloroadenosine greater than N6-(R)-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6-(S)-phenylisopropyladenosine. This potency order was the same for the binding of [3H]CGS 21680 to rat, and of [3H]NECA in the presence of 50 nM CPA to rat and human, brain membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of adenosine receptors in a model of cultured neurons from rat forebrain

The neuromodulator adenosine is acting through specific receptors coupled to adenylate cyclase via G-proteins. The expression of both adenosine receptors A₁ and A₂ as well as forkolin binding sites was investigated by radioligand binding techniques in 8-day-old neurons isolated from fetal rat forebrain and cultured in chemically-defined medium. Adenosine A₁ receptors were specifically labeled with [³H]chloro-N⁶-cyclopentyladenosine (CCPA), whereas [³H]CGS 21680 was used for the analysis of A₂ receptors. Cultured neurons exhibited high affinity binding sites for CCPA (Bmax=160 fmol/mg protein; Kd=2.9 nM), and for CGS 21680 (Bmax=14 fmol/mg protein; Kd=1.7 nM). These data correlate well with those obtained in crude membranes isolated from the newborn rat forebrain. The incubation of culture membranes in the additional presence of guanylyl-5-imidodiphosphate (Gpp(NH)p, a GTP analogue) led to significantly increased Kd-values, suggesting the association of adenosine receptors with G-proteins. Finally, cultured neurons also bound specifically [³H]forskolin with characteristics close to those found in the newborn brain, indicating that cultured neurons appear as an appropriate model for studying the neuromodulatory properties of adenosine.     

Evaluation of prostaglandin-receptors in human and rat liver: interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50:2.5 +/- 1.7, (6.1 +/- 2.1) microM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9 +/- 0.9 (2.0 +/- 0.8) microM. The Scatchard analysis on [3H]PGE1- as well as on [3H]iloprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3 +/- 5.2 (21.3 +/- 4.3) fmol/mg protein and a Kd of 2.1 +/- 1.8 (1.9 +/- 0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4 +/- 18.2 (86.1 +/- 13.2) fmol/mg protein and a Kd of 10.5 +/- 2.9 (15.1 +/- 3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4 +/- 13.9 (35.9 +/- 8.2) fmol/mg protein and a Kd of 4.1 +/- 1.2 (1.7 +/- 1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3 +/- 42.1 (142.9 +/- 17.8) fmol/mg protein and a Kd of 16.3 +/- 4.9 (9.2 +/- 7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4 +/- 51.9 (38.8 +/- 7.4) fmol/mg protein and a Kd of 16.2 +/- 3.2 (2.5 +/- 1.2) nM. It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are able to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.     

Calcium channel binding in nerves and muscle of canine small intestine

Using [3H]-nitrendipine (Nit) and [125I]-omega conotoxin (w-CTX), the cellular and subcellular distribution of calcium channel subtypes in the homogenates of canine small intestinal circular muscle was studied. Nit. bound to the membranes from the circular smooth muscle cells (PM) and to the synaptosomal membranes from the deep muscular plexus (DMP); the Kd and Bmax values of Nit binding from these two sources were similar (Kd 0.4 +/- 0.16 nM and 0.77 +/- 0.24 nM; Bmax 206 +/- 22 and 192 +/- 39 fmol/mg of protein in DMP and PM respectively). w-CTX, however, bound only to the DMP (Kd 18.41 +/- 7.5 pM, Bmax 265 +/- 36 fmol/mg of protein). In DMP, nifedipine (10(-6) M) failed to interact with the binding of w-CTX; similarly, no modulation of Nit binding with unlabelled w-CTX (10(-7) M) could be detected. Therefore w-CTX and Nit binding sites represent two distinct, non-interactive and differentially distributed binding sites in canine small intestine.     

19-hydroxyandrostenedione does not modulate [3H]aldosterone binding to human mononuclear leucocytes and rat renal cytosol

To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.     

Characterization of adenosine receptors in Mullus surmuletus

The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A₁ receptor selective agonist, [³H]CHA, and A_2a receptor selective agonist [³H]CGS 21680. The A₁ selective agonist, [³H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (K_d 1.47 nM; B_max 100–190 fmol/mg protein, dependent on fish length). The A_2a selective agonist, [³H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (K_d 44.2 nM; B_max 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [³H]CHA and [³H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [³H]CHA and [³H]CGS 21680 was inhibited by GDPβS. For [³H]CHA the IC₅₀ value was 2.5 ± 0.1 μM, while for [³H]CGS 21680 the IC₅₀ value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [³H]CHA have some pharmacological characteristics of mammalian A₁ adenosine receptors, while the binding sites for [³H]CGS 21680 appear to be virtually identical to the binding sites for [³H]CHA.     

Identification and characterization of leukotriene C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1

We studied the characteristics of the leukotriene (LT) C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1. Specific [3H]LTC4 binding to the cell membrane was greater than 80% of total binding and saturable at a density of 3.96 +/- 0.39 pmol/mg protein, with an apparent dissociation constant (Kd) of 14.3 +/- 2.0 nM (n = 9). The association and dissociation of [3H]LTC4 binding were rapid and apparent equilibrium conditions were established within 5 min. Calculated Kd value of [3H]LTC4 binding from the kinetic analysis was 9.9 nM. From the competition analysis, calculated Ki value of unlabeled LTC4 to compete for the specific binding of [3H]LTC4 was 9.2 nM and was in good agreement with the Kd value obtained from the Scatchard plots or kinetic analysis. The rank order of potency of the unlabeled competitors for competing specific [3H]LTC4 binding was LTC4 much greater than LTD4 greater than LTE4 greater than FPL-55712. The maximum number of binding sites (Bmax) of [3H]LTD4 in the membrane of BC3H-1 cell line was about 11 times lower than that of the [3H]LTC4. The calculated values of Kd and Bmax of [3H]LTD4 binding were 9.3 +/- 0.8 nM and 0.37 +/- 0.04 pmol/mg protein, respectively (n = 3). The rank order of potency or the unlabeled competitors for competing specific [3H]LTD4 binding was LTD4 = LTE4 greater than FPL-55712 much greater than LTC4. These findings demonstrate that BC3H-1 cell line possess both LTC4 and LTD4 receptors with a predominance of LTC4 receptors. Thus BC3H-1 cell line is a good model to study the regulation of LTC4 and LTD4 receptors.     

Evaluation of prostaglandin-receptors in human and rat liver: Interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50: 2.5±1.7, (6.1±2.1) μM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9±0.9 (2.0±0.8) μM. The Scatchard analysis on [3H]PGE1- as well as on [3H]ilioprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3±5.2 (21.3±4.3) fmol/mg protein and a Kd of 2.1±1.8 (1.9±0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4±18.2 (86.1±13.2) fmol/mg protein and a Kd of 10.5±2.9 (15.3±3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4±13.9 (35.9±8.2) fmol/mg protein and a Kd of 4.1±1.2 (1.7±1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3±42.1 (142.9±17.8) fmol/mg protein and a Kd of 16.3±4.9 (9.2±7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4±51.9 (38.8±7.4) fmol/mg protein and a Kd of 16.2±3.2 (2.5±1.2) nM.It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are stable to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Identification of a high-affinity receptor for interleukin-1 beta in rat brain

A single type of high-affinity binding sites for IL-1 beta was identified in the rat hypothalamus (Kd = 1.0 +/- 0.2 nM) and cerebral cortex (Kd = 1.3 +/- 0.2 nM), but not in the pituitary. The maximum binding capacity (Bmax) in the hypothalamus (Bmax = 75.4 +/- 10.8 fmol/mg protein) was 4 times greater than in the cerebral cortex (Bmax = 17.2 +/- 1.5 fmol/mg protein). Neither various neuropeptides nor IL-2 appeared to influence the binding of [125I]IL-1 beta to the hypothalamic membrane preparations. The potency of unlabeled IL-1 alpha to replace the binding of [125I]IL-1 beta to the hypothalamic membrane preparations was considerably less than that of unlabeled IL-1 beta. These findings indicate that IL-1 beta receptors are heterogeneously distributed in the central nervous system and that IL-1 alpha does not bind with IL-1 beta receptors in the brain.     

[3H]Dipyridamole Binding to Guinea Pig Brain Membranes: Possible Heterogeneity of Central Adenosine Uptake Sites

The binding of [3H]dipyridamole ([3H]DPR) to guinea pig brain membranes is described and compared to that of [3H]nitrobenzylthioinosine ([3H]NBI). The binding of [3H]DPR is saturable, reversible, and specific with pharmacologic evidence indicating that this ligand is binding to the adenosine uptake site. Compared to [3H]NBI the binding of [3H]DPR is of higher capacity (Bmax = 208 +/- 16 fmol/mg protein for [3H]NBI and 530 +/- 40 fmol/mg protein for [3H]DPR) and lower affinity (KD = 0.35 +/- 0.02 nM for [3H]NBI and 7.6 +/- 0.7 nM for [3H]DPR). The adenosine uptake inhibitors are the most potent inhibitors of binding (Ki of 10(-8)-10(-7) M) whereas adenosine receptor ligands such as cyclohexyladenosine, 2-chloroadenosine, and various methylxanthines are several orders of magnitude less potent (Ki 10(-5)-10(-2). The inhibition of [3H]DPR binding by NBI is biphasic, with only 40% of binding being susceptible to inhibition of NBI concentrations less than 10(-5) M. The tissue distribution of [3H]DPR binding parallels that of [3H]NBI although in most cases significantly more sites are observed with [3H]DPR. Calcium channel blocking agents such as nifedipine, nimodipine, and verapamil are also inhibitors of [3H]DPR binding with potencies in the micromolar range. The data are consistent with [3H]DPR being a useful additional ligand for the adenosine uptake site and provide evidence that multiple uptake binding sites exist of which only about 40% are NBI-sensitive.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

HPLC-purified 2-[125I]iodomelatonin labels multiple binding sites in hamster brain

Binding of 2-[125I]iodomelatonin in hamster brain synaptosomal membranes at 0 degrees C is rapid, saturable, reversible and sensitive to heat and trypsin treatment. Computer resolution of curvilinear Scatchard plots yielded high- and low-affinity components as follows: Kd1 = 0.32 +/- 0.14 nM, Bmax1 = 5.6 +/- 1.7 fmol/mg protein and Kd2 = 10.5 +/- 3.2 nM, Bmax2 = 123 +/- 33 fmol/mg protein (n = 3). Competition experiments indicated that 2-iodomelatonin and prazosin are the most potent inhibitors of high-affinity binding. Unlike prazosin, several alpha-adrenergic agents and various neurotransmitters were ineffective. These findings suggest that prazosin may be a potent antagonist at a unique, non-alpha-adrenergic, high-affinity binding site for melatonin.     

Synthesis and preliminary biological evaluation of [3H]-MRE 3008-F20: the first high affinity radioligand antagonist for the human A3 adenosine receptors

The synthesis and the preliminary biological evaluation of the first high affinity radioligand antagonist for the human A3 adenosine receptor, named [3H]-MRE 3008-F20 are reported. [3H]-MRE 3008-20 bound human A3 receptors expressed in CHO cells with K(D) and Bmax value of 0.82 +/- 0.08 nM and 297 +/- 28 fmol/mg of protein, respectively. [3H]-MRE 3008-F20 represents a useful tool for a further characterization of A3 adenosine receptor subtype.     

Magnesium-Dependent Enhancement of Endogenous Agonist Binding to A1 Adenosine Receptors: A Complicating Factor in Quantitative Autoradiography

Quantitative autoradiography was used to investigate the effects of Mg2+ on agonist and antagonist binding to A1 receptors in rat striatum. A1 receptors were labelled with the selective agonist N6-[3H]cyclohexyladenosine ([3H]CHA) or the selective antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine ([3H]DPCPX). Mg2+ had no significant effect on equilibrium binding constants for [3H]CHA [control: KD (95% confidence interval) of 0.34 (0.15-0.80) nM and Bmax of 267 +/- 8 fmol/mg of gray matter; with 10 mM Mg2+: KD of 0.8 (0.13-4.9) nM and Bmax of 313 +/- 8.9 fmol/mg of gray matter] or [3H]DPCPX [control: KD of 0.54 (0.30-0.99) nM and Bmax of 256 +/- 2.3 fmol/mg of gray matter; with 10 mM Mg2+: KD of 1.54 (0.2-11.0) nM and Bmax of 269 +/- 35.7 fmol/mg of gray matter]. In contrast, Mg2+ slowed the apparent association rate for both ligands; this was observed as a shift from a one-component to a two-component model for [3H]DPCPX. Mg2+ also affected the dissociation rates of both ligands; for [3H]CHA, dissociation in the presence of Mg2+ was not detected. Mg2+ produced a concentration-dependent inhibition of [3H]CHA binding only prior to equilibrium. HPLC was performed on untreated sections, sections preincubated with adenosine deaminase (ADA), and sections preincubated with ADA and incubated with ADA in the absence or presence of Mg2+. Adenosine was found in measurable quantities under all conditions, and the concentration was not influenced by Mg2+ or by the inclusion of GTP in the preincubation medium. From these data, we conclude the following: (a) adenosine is present and may be produced continuously in brain sections; (b) ADA is not capable of completely eliminating the produced adenosine; (c) Mg2+ apparently does not influence adenosine production or elimination; (d) A1 receptor-guanine nucleotide binding protein coupling is maximal in this preparation; and (e) Mg2+ decreases the dissociation rate of bound endogenous adenosine from A1 receptors, thus limiting the access of [3H]CHA and [3H]DPCPX to the receptors. Thus, enhancement of endogenous adenosine binding to A1 receptors by Mg2+ is a complicating factor in receptor autoradiography and may be so in other preparations as well.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Binding of the hydrophilic beta-adrenergic antagonist [3H]CGP-12177 to cardiac tissue slices: characterization and ontogenetic studies in dogs

A new technique was developed to characterize the binding of a hydrophilic beta-adrenergic antagonist, [3H]CGP-12177, to 1-mm thick slices of canine cardiac tissue. This technique was used to quantify the density (Bmax) and the affinity (Kd) of these receptors in the right ventricular conus (RVC) and the left ventricle (LV) at day 1 to 6 weeks of age, and in the adult. Binding was found to be reversible, saturable, stereospecific, of high affinity, and thermolabile. There was an increase in the density of beta-adrenergic receptors between day 1 (Bmax = 2.2 +/- 0.3 fmol/mg tissue in RVC and 2.9 +/- 0.8 fmol/mg tissue in the LV) and 2 weeks of age postnatally, after which it remained constant until 6 weeks of age (Bmax = 7.5 +/- 0.4 and 6.8 +/- 0.9 fmol/mg tissue in RVC and LV, respectively); however, by 6 weeks of age it had not reached adult levels (10.3 +/- 1.0 fmol/mg tissue). The affinity of these receptors did not change between early neonatal life (Kd = 1.3 +/- 0.4 nM) and adulthood (Kd = 1.4 +/- 0.2 nM). The density of beta-adrenergic receptors in the RVC was similar to that in the LV. This new method of quantifying beta-adrenergic receptors in cardiac tissue is simple and fast, and requires minimal tissue handling. It proved to be useful in studying the development of cardiac beta-adrenergic receptors with age.     

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.     

(+)-PN200-l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitrobenzylthioinosine binding in brain: an interspecies study

The binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ( [3H]NBI) to cerebral cortical membrane preparations from human, dog, guinea pig, rat, and mouse was investigated. Reversible, specific, saturable, high affinity binding was found in all five species with similar kinetic parameters. (Kd = 0.16-0.44 nM; Bmax = 128-196 fmol/mg prot.). Dilazep, hexobendine, and dipyridamole were all high potency inhibitors of [3H]NBI binding in human, dog, guinea pig and mouse preparations but not in rat. These compounds showed a competitive inhibition of [3H]NBI binding indicating that they are acting at the same site. Discrepancies seen in the rat appear to be a unique, species related anomaly. The dihydropyridine calcium antagonists also inhibited binding with lower potency than the adenosine uptake blockers. This inhibition was most potent in dog and human and suggests a relationship between the calcium channel and the adenosine uptake site.