Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.     

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.     

beta-Adrenergic receptors in rat brain.

125I-Iodohydroxybenzylpindolol ([125I] IHYP), a potent beta-adrenergic receptor antagonist, has been used to study beta-adrenergic receptors in rat brain. Binding of [125I] IHYP (30 pM) to a membrane fraction min and dissociation took place with a half time of about 16 min. Phentolamine (10(-4) M) decreased non-receptor binding but it had no effect on the binding of [125I] IHYP to beta-adrenergic receptors in cortex, cerebellum or caudate. In the presence of phentolamine specific binding (defined as binding which was blocked by 0.3 muM dl-propranolol) represented 70-85% of total binding. The binding of [125I] IHYP was inhibited by beta-adrenergic agonists and antagonists. d-Stereoisomers were 2-3 orders of magnitude less potent than the corresponding 1-isomers. The denstiy of [125I] IHYP binding sites was studied in membrane fractions from cerebral cortex, cerebellum, and caudate nucleus by means of Scatchard analysis. The K(D) of [125I] IHYP was similar in the three regions studied, and the density of [125I] IHYP binding sites was approximately 50% greater in the cortex and caudate than in the cerebellum. The Hill coefficient for the binding of [125I] IHYP to membranes from cerebral cortex was 1.02. The properties of the binding of [125I] IHYP are similar to those which would be expected of binding to beta-adrenergic receptors in vitro.     

Adrenergic receptors in the liver parenchyma in children with chronic hepatitis]

In the present study adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of extrahepatic portal hypertension children without parenchymal affection (control group, n-7) and the resection of children in parenchymal affection (group of chronic hepatitis children, n-6). It has been shown, that the binding of beta-adrenergic radioligand 3H-dihydroalprenolol (3H-DHA) in liver parenchyma membranes of both control and chronic hepatitis groups was saturable and showed high affinity. The Scatchard analysis of the binding data indicated that the binding site was characterized by Kd and Bmax of 1.2 +/- 0.5 nM, 261.2 +/- 50 fmol/mg, respectively, for the control group; and 0.9 +/- 0.15 nM, 68.5 +/- 18.8 fmol/mg, respectively, for the group of chronic hepatitis patients; (mean+SEM). The binding of alpha 1-adrenergic antagonist 3H-prazosin (3H-PRZ) in liver parenchyma was also saturable and showed high affinity. The binding site is characterized by Kd = 0.6 +/- 0.12 nM, Bmax = 92.8 +/- 8.0 fmol/mg, for the control group; and Kd = 0.8 +/- 0.15 nM, Bmax = 195.0 +/- 22.0 fmol/mg, for the group of chronic hepatitis. It has been found that the number of binding sites of 3H-DHA significantly decreased and the number of binding sites of 3H-PRZ did not change in chronic hepatitis liver parenchyma in comparison with the control group. The results obtained suggest the important role of beta-adrenergic receptors in the pathogenesis of chronic hepatitis and in liver regeneration in children.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of atrial natriuretic factor receptors in the rat retina

The characteristics of atrial natriuretic factor (ANF) receptors where studied in rat retinal particulate preparations. Specific 125I-ANF binding to retinal particulate preparations was greater than 90% of total binding and saturable at a density (Bmax) of 40 +/- 8 fmol/mg protein with an apparent dissociation constant (Kd) of 6.0 +/- 2.0 pM (n = 3). Apparent equilibrium conditions were established within 30 min. The Kd value of 125I-ANF binding calculated by kinetic analysis was 4.0 pM. The Bmax of 60 +/- 10 fmol/mg protein and the Kd of 5 +/- 2 pM, calculated by competition analysis, were in close agreement with the values obtained from Scatchard plots or kinetic analysis. The 125I-ANF binding to retinal particulate preparations was not inhibited by 1 microM concentration of somatostatin, vasopressin, vasoactive intestinal peptide, adrenocorticotropin, thyrotropin releasing hormone, or leu-enkephalin. The rank order of potency of the unlabelled atrial natriuretic peptides for competing with specific 125I-ANF (101-126) binding sites was rANF (92-126) greater than rANF (101-126) greater than rANF (99-126) greater than rANF (103-126) greater than Tyro-Atriopeptin I greater than hANF (105-126) greater than rANF (1-126). Similar results have been obtained in peripheral tissues and mammalian brain, indicating that central and peripheral ANF-binding sites have somewhat similar structural requirements. Affinity cross-linking of 125I-ANF to retinal particulate preparations resulted in the labelling of two sites of molecular weight 140 and 66 kDa, respectively. This demonstration of specific high-affinity ANF receptors suggests that the peptide may act as a neurotransmitter or neuromodulator in the retina.     

High density of endothelin binding sites in the hearts of infants and children

Endothelin (ET)-1 peripheral levels are high in children with respect to values of adults, but its pathophysiological significance remains to be established. In these conditions the interaction of ET-1 with its receptors may constitute a clue to the understanding of ET-1 function. Because a direct determination of ET binding sites in the heart of children is lacking, in this study we have attempted an assessment of the ET receptor status in cardiac tissue of infants (<1 year; 0.39 +/- 0.26 (SD) years, n=6) and children (1-14 years; 6.3 +/- 4.9 years, n=7) as well as an evaluation of the receptor modulation as a function of age, associated to the observed decrease of plasma ET levels between infants and children. ET-1 binding sites have also been evaluated in atrium and ventricle membranes of adult subjects recipient of cardiac transplantation (CHF) and of post-mortem cardiac specimens (autopsy) of non cardiac patients. Considering all the pediatric patients (infants +/- children) studied, an affinity constant (Kd) value of 38.2 +/- 6.1 (SEM) pM and a density (Bmax) value of 166.2 +/- 11.6 fmol/mg protein has been obtained for atrium. Similar values have been found in the ventricle. These values are significantly higher with respect to those obtained in adults: for atrial membranes, Kd = 22.2 +/- 9.7 and 11.6 +/- 1.8 pM; Bmax = 58.4 +/- 22.8 and 42.1 +/- 8.9 fmol/mg protein, respectively in explanted hearts and in post mortem specimens. No significant differences have been found in the binding parameters between infants and children, while, considering our results as a whole, a significant inverse correlation between Bmax and subject age (p<0.001) is suggested. The ET-A/ET-B ratio, evaluated by competition experiments with the specific ET-A antagonist BQ-123, was about 70:30 in pediatric patients, in both atrium and ventricle, without any difference between infants and children. Similar values for ET-A/ET-B ratio in adult CHF patients, in contrast to a reduction (significant only in ventricle) of the percent of ET-A subtype in autopsy, has been found. This is the first study concerning a direct evaluation of ET receptor status in children's hearts; the higher density of binding sites, associated to the elevation of plasma levels, could suggest a enhanced biological function of ET in children.     

Beta-adrenergic receptors and myogenesis.

The rat myogenic cell line, L8, contains a beta-adrenergic catecholamine-sensitive adenylate cyclase. Prior to cell fusion, and continuing thereafter, beta-adrenergic sites, as determined by the stereospecific binding of (125I)-hydroxybenqylpindolol, I1(125I)IHYP] increases from 470 to 2000 sites/cell. There is also an increase in adenylate cyclase (2-5 fold) and endogenous cAMP (5-30 fold) following stimulation by catecholamine. The dissociation constant (KD) of (125I)IHYP for unfused and fused cell-homogenates, as determined by estimation with Scatchard analysis, by direct determination at receptor concentrations well below the KD, or by association (4.6 X 10(8) M-1 min-1); and dissociation (0.028 min-1) kinetics; ranged from about 40 to 70 pM. The acquisition of beta-receptors prior to fusion in L8 cells may implicate this system in the regulation of myogenesis.     

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in syrian hamster hypothalamus

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.     

Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Identification and characterization of melatonin binding sites in the gastrointestinal tract of ducks.

Utilizing 2-[125I]iodomelatonin as the radioligand, melatonin binding sites were identified and characterized in the jejunum of ducks. These sites were found to be reversible, saturable, specific and exhibited high affinity for melatonin. Scatchard analyses have established the equilibrium dissociation constant (Kd) for tissues collected during mid-photophase to be 40.9 +/- 7 pM and the maximum quantity of binding sites (Bmax) to be 2.0 +/- 0.4 fmol/mg protein while Kd of samples collected during mid-scotophase was found to be 54.1 +/- 10 pM with a corresponding Bmax of 1.5 +/- 0.3 fmol/mg protein. These Kd values are in good proximity to the kinetically derived equilibrium dissociation constant of 47.3 +/- 20 pM. No significant difference in Kd or Bmax was detected between the mid-light and mid-dark samples. Pharmacological profile of these binding sites, developed by their interactions with other indoles and compounds, indicated that these binding sites are highly specific for melatonin. Subcellularly, different densities of binding sites were localized to various fractions in the following order: nuclear greater than microsomal greater than mitochondrial greater than cytosolic. These binding sites in the jejunum might be the receptors accountable for promoting paracrine activities for the locally synthesized gastrointestinal melatonin and/or responsible for eliciting hormonal actions via interactions with melatonin of pineal origin.     

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.     

Photoaffinity labeling of the beta-adrenergic receptor with azide derivatives of iodoccyanopindolol

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.     

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.     

Absence of age-related changes in the binding of the beta adrenergic antagonist 125I-iodohydroxybenzylpindolol in rat heart

The effect of age on the density and the affinity of beta adrenergic receptors was determined in the hearts of Fischer 344 rats at three ages, 6, 12, and 24 months old. The binding of the beta adrenergic antagonist 125I-iodohydroxybenzylpindolol (IHYP), was used to quantitate and characterize cardiac beta adrenergic receptors. The maximal number of binding sites (Bmax = F moles/mg of protein) were 26.3 +/- 2.5, 25.4 +/- 0.99, and 24.5 +/- 2.4 and the dissociation constants (Kd = nM) were 0.166 +/- 0.014, 0.126 +/- 0.006, and 0.135 +/- 0.015 for 6, 12, and 24 months old animals, respectively. There were no significant differences among the three ages. These results support the contention that neither beta adrenergic receptor density or affinity changes with age in the ventricles of the rat heart.     

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.     

Identification and characterization of cellular receptors for the growth regulator, oncostatin M

Oncostatin M is a polypeptide growth regulator produced by activated T cells and phorbol ester-treated U937 cells. To identify specific cellular receptors for this factor, we have characterized the binding of 125I-labeled oncostatin M to a variety of normal and malignant mammalian cells. Recombinant oncostatin M was labeled with 125I with full retention of growth inhibitory activity on A375 melanoma cells. 125I-Oncostatin M bound to sensitive cells in a time- and temperature-dependent fashion. Binding was specifically inhibited by unlabeled native or recombinant oncostatin M, but not by other polypeptide growth factors tested. Binding to human leukemic and normal blood cells was generally less than to nonhematopoietic cells. With four different cell lines, maximal growth inhibition by oncostatin M was achieved at less than maximal binding site occupancy. Scatchard graphs of direct binding data were curvilinear and indicated that 125I-oncostatin M bound with higher apparent affinity at lower 125I-oncostatin M concentrations. Using a two binding site model, affinity constants of Kd1 = 11 +/- 11 pM and Kd2 = 1000 +/- 380 pM were extrapolated from binding data with A375 cells, and values of Kd1 = 3 +/- 2 pM and Kd2 = 400 +/- 44 pM from A549 cells. The major 125I-oncostatin M binding species in a number of mammalian cell lines was identified by chemical cross-linking as a specific protein(s) of Mr = 150,000-160,000. 125I-Oncostatin M was internalized (t1/2 = 30 min) and degraded subsequent to binding to a responsive cell line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin II binding sites in frog kidney and adrenal.

Angiotensin II (AII) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. AII binding was present in kidney glomeruli and in interrenal tissue of the outer zone of the adrenal gland. Saturation experiments showed that [125I]-[Val5]AII binds to a single class of binding sites with a dissociation constant (Kd) of 548 +/- 125 pM in glomeruli and 593 +/- 185 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 2.48 +/- 0.71 and 3.05 +/- 1.02 fmol/mm2, respectively. AII binding was displaced by unlabeled angiotensin analogues in the rank order: [Sar1]AII greater than human AII greater than [125I]-[Val5]AII = [Val5]AII = human AIII much greater than human AI. The AII binding sites in glomeruli and interrenal tissue suggest an influence of AII on glomerular filtration rate and adrenal steroid secretion to take part in osmomineral regulation of the frog.