Alpha and beta adrenergic receptors of canine lung tissue identification and characterization of alpha adrenergic receptors by two different ligands

Adrenergic receptors of canine peripheral lung tissues were measured by direct binding techniques using [³H]dihydroergocryptine ([³H]DHE), [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA). All three ligands bound to canine lung tissue with saturability, stereospecificity and reversibility. Adrenergic agonists competed for binding of [³H]DHE and [³H]prazosin in the order: 1-epinephrine > 1-norepinephrine > d-epinephrine > d-norepinephrine > 1-isoproterenol. Adrenergic antagonists competed for binding of [³H]prazosin in the order: prazosin > phentolamine > yohimbine. Inhibition curves of [³H]DHE by prazosin or yohimbine were biphasic suggesting two subtypes of binding sites. Maximum binding capacities of [³H]DHE ranged from 30.6 to 42.7 fmol/mg protein. [³H]prazosin from 18.3 to 26.9 fmol/mg protein and [³H]DHA from 135.2 to 359.4 fmol/mg protein. When both [³H]DHE and [³H]prazosin were used in the same membrane preparation, specific binding of [³H]DHE was always more than that of [³H]prazosin. Since [³H]prazosin is considered to bind to alpha₁ adrenergic receptors specifically and [³H]DHE is considered to bind alpha₂ adrenergic receptors nonselectively, the difference between the numbers of the specific binding sites of these two ligands should represent alpha₂ adrenergic receptors. Alpha₂ adrenergic receptor density ranged from 9.5 to 21.1 fmol/mg protein. Our results suggest the existence of both alpha₁ and alpha₂ adrenergic receptors in canine peripheral lung tissue. Approximately 40% of alpha adrenergic receptors were alpha₂. The ratio of alpha/beta adrenrgic receptors ranged from 1:3.3 to 1:10.4. The ratio of alpha₁/be ta adrenergic receptors ranged from 1:6.7 to 1:21.1.     

Lack of specific (3H) prazosin binding sites in dog and rabbit cerebral arteries

In order to explore the characteristics of alpha adrenergic receptors on cerebrovascular smooth muscle, specific binding sites for the alpha 1 adrenergic ligand, (3H) prazosin, were studied in blood vessel homogenates. No specific (3H) prazosin binding was found in either rabbit or dog cerebral arteries, but specific binding was demonstrated in the rabbit saphenous and ear arteries. In the ear artery 3H-prazosin binding was saturable with a Kd of 0.51 +/- 0.20 nM and a Bmax of 89 +/- 29 fmoles/mg protein. To confirm the adequacy of our membrane preparation, homogenates of both dog and rabbit cerebral arteries showed saturable specific binding with two different ligands: one for muscarinic receptors, [3H](-) quinuclidinyl benzilate (QNB) and one for alpha 2 adrenergic receptors, (3H) yohimbine. The results of these studies demonstrate a lack of alpha 1 adrenergic receptors on cerebral blood vessels, confirming functional studies showing only a weak contractile response to norepinephrine.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

Identification of alpha 1 adrenergic receptors in rabbit aorta with [125I] BE2254

Alpha-adrenergic receptors may play an important role in regulating vascular tone and reactivity. To study alpha-adrenergic receptors in blood vessels, we have developed a method to characterize and quantitate alpha-adrenergic receptors in a particulate fraction of individual rabbit aortas using the high specific activity alpha antagonist [125I] BE2254. [125I] BE2254 specifically labels a single class of binding sites with a dissociation constant of 286 pM and a maximal binding capacity of 16.7 fmoles/mg protein. Catecholamines compete for [125I] BE2254 binding stereospecifically and with the characteristic alpha-adrenergic potency series of (-)epinephrine greater than or equal to (-)norepinephrine much greater than (-)isoproterenol. The alpha 1-selective antagonist prazosin (KD = 0.7 nM) is much more potent in competing for [125I] BE2254 binding than is the alpha 2-selective antagonist yohimbine (KD = 1000 nM), which suggests that the alpha adrenergic receptor identified is predominantly of the alpha 1 subtype. Also, the dissociation constants from these binding studies were in good agreement with those reported in rabbit aorta from classical pharmacological experiments where contraction was found to be mediated via alpha 1 receptors. This extension of radioligand binding techniques to individual rabbit aortas should simplify the study of vascular alpha adrenergic receptor regulation, and provide a basis for broadening the understanding of vascular alpha adrenergic receptors.     

Human apolipoprotein A-II: nucleotide sequence of a cloned cDNA, and localization of its structural gene on human chromosome 1

[3H]yohimbine, a potent and selective alpha 2-adrenergic antagonist was used to label alpha-adrenoceptors in intact human lymphocytes. Binding of [3H]yohimbine was rapid (t1/2 1.5 -2.0 min) and readily reversed by 10 microM phentolamine (t1/2 = 5 - 6 min) and of high affinity (Kd = 3.7 +/- 0.86 nM). At saturation, the total number of binding sites was 19.9 +/- 5.3 fmol/10(7) lymphocytes. Adrenergic agonists competed for [3H]yohimbine binding sites with an order of potency: clonidine greater than (-) epinephrine greater than (-) norepinephrine greater than (+) epinephrine much greater than (-) isoproterenol; adrenergic antagonists with a potency order of yohimbine greater than phentolamine greater than prazosin. These results indicate the presence of alpha 2-adrenoceptors in human lymphocytes.     

Subcellular distribution of alpha 1-adrenergic receptors in rat liver

The distribution of alpha 1-adrenergic receptors in rat liver subcellular fractions was studied using the alpha 1-adrenergic receptor ligand [3H]prazosin. The highest number of [3H]prazosin binding sites was found in a plasma membrane fraction followed by 2 Golgi and a residual microsomal fraction, the numbers of binding sites were 1145, 845, 629 and 223 fmol/mg protein, respectively. When the binding in these fractions was compared with the activity of plasma membrane 'marker' enzymes in the same fractions a relative enrichment of [3H]prazosin binding sites was found in the residual microsomes and one of the Golgi fractions. Photoaffinity labelling with 125I-arylazidoprazosin in combination with SDS-polyacrylamide gel electrophoresis revealed the specific binding to 40 and 23 kDa entities in a Golgi fraction, while in plasma membranes the binders had an apparent molecular mass of 36 and 23 kDa. When [3H]prazosin was injected in vivo into rat portal blood followed by subcellular fractionation of liver, a pattern of an initial rapid decline and thereafter a slow decline of radioactivity was noted in all fractions. Additionally, in the two Golgi fractions a transient accumulation of radioactivity occurred between 5 and 10 min after the injection. The ED50 values for displacement of [3H]prazosin with adrenaline was lowest in the plasma membrane fraction, followed by the residual microsomes and Golgi fractions, the values were 10(-6), 10(-5) and 10(-4) mol/l, respectively. On the basis of lack of correlation between distribution of alpha 1-adrenergic antagonist binding and adenylate cyclase activity, differences in the molecular mass of alpha 1-adrenergic antagonist binders, differences in the kinetics of in vivo binding and accumulation of [3H]prazosin and also differences in agonist affinity between plasma membrane and Golgi fractions, it is concluded that alpha 1-adrenergic receptors are localized to low-density intracellular membranes involved in receptor biosynthesis and endocytosis.     

Involvement of norepinephrine activity in the regulation of alpha 1 adrenergic receptors in the medial preoptic nucleus of estradiol-treated rats

To establish whether the diurnal decrease in the density of alpha 1 receptors observed in the medial preoptic nucleus (MPN) of estrogen (E2)-treated rats is related to the concomitant diurnal increase in norepinephrine (NE) turnover rates, we quantitated the density of [3H]-Prazosin binding to alpha 1 receptors after blockade of NE turnover with alpha-methyl-paratyrosine (alpha MPT). A series of preliminary studies was performed to rule out an interference of this drug with [3H]-Prazosin binding to alpha 1 adrenergic receptors in vitro and in vivo. Incubation of brain slices with alpha MPT produced a dose-dependent inhibition of [3H]-Prazosin binding to alpha 1 adrenergic receptors with an IC50 of approximately 6 mM. Scatchard analysis demonstrated that alpha MPT exhibited a simple competitive interaction with [3H]-Prazosin binding sites as shown by an increase in the apparent dissociation constant (Kd) of the ligand and no change in the number of alpha 1 receptors (Bmax). In contrast, preincubation of brain slices with alpha MPT and prior in vivo administration of alpha MPT did not affect [3H]-Prazosin binding to alpha 1 adrenergic receptors. Once we established that alpha MPT could be used to suppress NE turnover without interfering with the measurement of alpha 1 receptor densities, we repeatedly injected this drug to ovariectomized (OVX) and E2-implanted rats. The density of alpha 1 adrenergic receptors in MPN was quantitated autoradiographically. Blockade of NE turnover with alpha MPT only partially prevented the reduction in alpha 1 receptor density observed in the E2-treated rats, suggesting that the decrease in the level of [3H]-Prazosin binding sites cannot be completely ascribed to increased NE turnover rates.     

Comparison of muscarinic and beta adrenergic receptors between bovine peripheral lung and tracheal smooth muscles: a striking difference in the receptor concentration

This study was undertaken to compare the activity of muscarinic and beta adrenergic receptors in bovine peripheral lung to the corresponding receptor activity in tracheal smooth muscle. We used [3H] quinuclidinyl benzilate (QNB) and [3H]dihydroalprenolol (DHA) to measure muscarinic and beta receptor activity, respectively. Binding to QNB and DHA at 25 degrees C was rapid, reversible, saturable and of high affinity. The order of potency for cholinergic and adrenergic agents competing for binding was compatible with muscarinic and beta 2 adrenergic potencies. We found that the concentration of muscarinic receptor binding sites was 37-fold greater in the tracheal muscle preparation (2805 +/- 309 fmol/mg protein) than in the peripheral lung preparation (76 +/- 28 fmol/mg protein). Unlike muscarinic receptors, the lung contained 8-fold higher concentration of the beta adrenergic receptors than did the tracheal muscle (1588 +/- 417 vs. 199 +/- 42 fmol/mg protein). The dissociation constant or the agonist's inhibitory constant (Ki) for either receptor binding site, however, was not significantly different between the two tissues. Furthermore, in vitro contraction studies showed that the response of tracheal muscle strips to methacholine was markedly greater than the response of peripheral lung strips, a finding consistent with the QNB binding result. The muscle but not the peripheral lung strip exhibited a relaxing response to epinephrine. Our data indicate a striking quantitative difference in muscarinic and beta adrenergic receptors between lung tissue and tracheal muscle, and that each receptor in the lung is qualitatively similar to the corresponding receptor in the muscle.     

Identification and characterization of (3H)-rauwolscine binding to alpha 2-adrenoceptors in the canine saphenous vein

The biochemical exploration of the alpha 2-adrenergic receptors was investigated in the canine saphenous vein using the highly selective alpha 2-adrenergic antagonist rauwolscine as a tritiated ligand. Following an enzymatic digestive pretreatment, we isolated a purified smooth muscle cell membrane fraction from saphenous veins in quantity sufficient to permit us to study the venous alpha 2-adrenoceptor content. The binding of tritiated rauwolscine was rapid, specific, saturable and reversible. The presence of high affinity binding sites (Kd = 1.53 +/- 0.71 nM) with a density of binding Bmax of 125.2 +/- 43.1 fmol/mg protein was demonstrated on a unique class of non interacting sites (nHill = 1.001 +/- 0.06). The kinetically derived Kd was 1.28 nM, in good agreement with the value obtained from saturation isotherms. The pharmacological profile of these sites was assessed by the comparison of the potency of alpha-adrenergic agonists and antagonists to inhibit 1 nM (3H)-rauwolscine. Their efficacy was respectively: rauwolscine greater than phentolamine greater than RX 781094 greater than clonidine much greater than prazosin greater than (-)-phenylephrine greater than (-)-noradrenaline. The results showed that (3H)-rauwolscine bound specifically to sites in our membranal preparation, which had the pharmacological characteristics of the alpha 2-adrenoceptors. The correlation between biochemical and pharmacological data revealed the usefulness of binding methods in the further study of adrenergic mechanisms in the canine saphenous vein.     

Identification of α2-Adrenergic Receptors in Chicken Pineal Gland Using [3H]Rauwolscine

The norepinephrine-induced inhibition of avian pineal N-acetyltransferase activity appears to be mediated by alpha 2-adrenergic receptors. In this study, alpha 2-adrenergic receptors in the chicken pineal gland were directly identified by radioligand binding. Membrane preparations of pineal glands from chickens from 1 to 6 weeks of age were examined using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, to characterize the binding sites. The results indicate no ontological change in either the affinity (KD) or density of receptor binding sites (Bmax) during the time span examined. The binding was saturable and of high affinity with a mean KD of 0.27 +/- 0.01 nM and a mean Bmax of 242 +/- 12 fmol/mg protein. Further characterization of these binding sites indicated that the alpha 2-adrenergic receptor is of the alpha 2A subtype, since prazosin and ARC-239 bound with low affinities and oxymetazoline bound with high affinity.     

Solubilization of Active Brain α1-Adrenoceptors by a Zwitterionic Detergent

Solubilization of rat brain alpha 1-adrenoceptors was performed by treatment with 6 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio] - 1 - propanesulfonate). The alpha 1-adrenoceptor antagonist [3H]prazosin was shown to bind reversibly and specifically to the soluble extract obtained after centrifugation at 150,000 X g for 1 h. Separation of the soluble [3H]prazosin-bound complexes was performed by the polyethylene glycol precipitation technique followed by filtration. A Scatchard plot of the concentration-dependent binding curve showed only one class of binding sites, with a high affinity for [3H]prazosin. Affinity of the solubilized receptors for the ligand increased as the CHAPS concentration in the assay medium decreased; the number of binding sites remained unchanged (approximately equal to 70 fmol/mg protein). This corresponds to a 30% recovery of original membrane sites. The solubilized receptors presented the same characteristics of specificity and stereospecificity as membrane alpha 1-adrenoceptors. Moreover, 150 mM NaCl was found to modulate the affinity of epinephrine for the [3H]prazosin-bound soluble complex, as previously described for membrane preparations. Thus, CHAPS appears to be a suitable detergent for solubilizing rat brain alpha 1-adrenoceptors and preserving their functional activities.     

Turnover of Adrenergic Receptors Under Normal and Desensitized Conditions

Abstract

Alpha₁ and beta adrenergic receptor metabolism was investigated by studying receptor reappearance after an irreversible blockade. Phenoxybenzamine was used to irreversibly block alpha₁ adrenergic receptors both in vitro in the BC₃H₁ cell line and in vivo in rat submaxillary glands. In these two systems, the alpha₁ adrenergic receptor reappearance followed a monoexponential kinetic allowing to determine the half-life of the receptor (23h in vitro, 33h in vivo) as well as the rate of receptor synthesis and degradation. the receptor reappearance was due to receptor synthesis since it was blocked by cycloheximide. The irreversible blockade of beta adrenergic receptors was done with an alkylating beta adrenergic antagonist that we recently developped : Br-pindolol (1). This ligand has high efficiency and blocked at 10^?7 M 80-90% of the beta adrenergic receptors present in C₆ glioma cells in culture. After this irreversible blockade, receptors reappeared only during cell division. At confluency, when cells did not significantly divide, receptor synthesis could hardly be detectable. Therefore, at confluency, the metabolic stability of the beta adrenergic receptor is considerable, compared to that of the alpha₁ adrenergic receptor. This stability was confirmed by the observation that after an almost complete “down-regulation” of the beta adrenergic receptor, receptor repopulation of the C₆ glioma cells was total and occured in the presence of cycloheximide.     

Beta adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation

C6 glioma cells possess beta adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br-AAM-pindolol), a beta adrenergic antagonist. With 1 microM Br-AAM-pindolol, more than 80% of beta adrenergic receptors, labeled by (3H)-dihydroalprenolol [3H)-DHA), were blocked. After this blockade, new beta adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of beta adrenergic receptors was barely detectable. Cycloheximide (1 microgram/ml) inhibited cell growth as well as reappearance of beta adrenergic receptors. A 90% loss of beta adrenergic receptors in C6 glioma cells was obtained after down-regulation for 15 h with 10 microM isoproterenol, a beta adrenergic agonist. After removal of the agonist, recovery of beta-adrenergic-sensitive adenylate cyclase was complete within 2 to 3 days, whereas beta adrenergic receptors reached 90% of control value within 6 days. The half-life of the receptor recovery was 2 to 3 days. Pretreatment of C6 glioma cells by Br-AAM-pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked beta adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of beta adrenergic receptors. The recovery of both biological response and beta adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 microgram/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of beta adrenergic receptors in C6 glioma cells, following isoproterenol-induced down regulation, was not due to synthesis of new receptors but to recycling of the beta adrenergic receptors.     

Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.     

The alpha 1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca(2+)-ATPase.

Membrane Ca(2+)-ATPase activity was stimulated in vitro separately by T4 (10(-10) M) and by epinephrine (10(-6) M). In the presence of a fixed concentration of T4, additions of 10(-8) and 10(-6) M epinephrine reduced the T4 effect on the enzyme. beta-Adrenergic blockade with propranolol (10(-6) M) prevented stimulation by epinephrine of Ca(2+)-ATPase activity, but did not prevent the suppressive action of epinephrine on T4-stimulable Ca(2+)-ATPase. In contrast, alpha 1-adrenergic blockade with unlabelled prazosin restored the effect of T4 on Ca(2+)-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulation by T4 of red cell Ca(2+)-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (Kd, 1.2 x 10(-8) M; Bmax, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains alpha 1-adrenergic receptor sites that are capable of regulating Ca(2+)-ATPase activity.     

Identification of alpha1-adrenergic receptors and their involvement in phosphoinositide hydrolysis in the frog heart

The aim of this study was to characterize alpha(1)-adrenergic receptors in frog heart and to examine their related signal transduction pathway. alpha(1)-Adrenergic binding sites were studied in purified heart membranes using the specific alpha(1)-adrenergic antagonist [(3)H]prazosin. Analysis of the binding data indicated one class of binding sites displaying a K(d) of 4.19 +/- 0.56 nM and a B(max) of 14.66 +/- 1.61 fmol/mg original wet weight. Adrenaline, noradrenaline, or phenylephrine, in the presence of propranolol, competed with [(3)H]prazosin binding with a similar potency and a K(i) value of about 10 microM. The kinetics of adrenaline binding was closely related to its biological effect. Adrenaline concentration dependently increased the production of inositol phosphates in the heart in the presence or absence of propranolol. Maximal stimulation was about 8.5-fold, and the half-maximum effective concentration was 30 and 21 microM in the absence and presence of propranolol, respectively. These data clearly show that alpha(1)-adrenergic receptors are coupled to the phosphoinositide hydrolysis in frog heart. To our knowledge, this is the first direct evidence supporting the presence of functional alpha(1)-adrenergic receptors in the frog heart.     

Ontogeny of alpha 1- and beta-adrenergic receptors in rat lung

The binding characteristics of the alpha 1-selective adrenergic ligand [3H]-prazosin were determined in particulate membranes of rat lung from day 18 of gestation to adulthood. Specific binding was present at all ages studied, was reversible and inhibition of specific binding by agonists followed the order of potency: (-)-epinephrine = (-)-norepinephrine much greater than (-)-isoproterenol greater than (+)-norepinephrine. Inhibition by antagonists followed the order of potency: prazosin greater than WB4101, much greater than yohimbine. Binding capacity increased during the neonatal period from 52 +/- 9 fmoles x mg-1 protein in lung preparations on day 18 of a 21 day gestation increasing to 105 +/- 4 fmoles x mg-1 protein (mean +/- SE) by postnatal day 15. Binding activity decreased thereafter, reaching adult levels by 28 days of postnatal age, 62 +/- 3 fmoles x mg-1 protein. This pattern of alpha 1-adrenergic receptor density was distinct from that of beta-adrenergic receptors identified in rat lung membrane with the beta- adrenergic antagonist, (-)-[3H]dihydroalprenolol ((-)-[3H]DHA). (-)-[3H]DHA binding increased dramatically during this same time period, from 46 +/- 4 fmoles x mg-1 protein on day 18 of gestation to 496 +/- 44 fmoles x mg-1 protein in the adult lung. Affinity for [3H]-prazosin and (-)-[3H]DHA did not change with age. Pulmonary alpha 1-adrenergic receptors are present as early as 18 days of gestation in the rat and alpha 1-adrenergic receptor density is maximal by 15 days of postnatal age. The timing of the changes in alpha 1-adrenergic receptors correlates with the timing of increased sympathetic innervation of the developing rat lung and is distinct from that of beta-adrenergic receptor sites.     

High level of expression of functional human platelet alpha 2-adrenergic receptors in a stable mouse C127 cell line

We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human alpha 2-adrenergic receptor gene. The size of the mRNA produced by the cloned gene is 1.8 kb. Electrophoretic analysis and autoradiography of cell membrane proteins photoaffinity labeled with p-[3H]azidoclonidine gave a broad protein band of molecular mass of approx. 64 kDa. Saturation binding with [3H]rauwolscine as ligand gave an equilibrium dissociation constant of 1.29 +/- 0.46 nM (mean +/- S.D.) and binding capacity range of 18-35 pmol/mg membrane protein, with (3-6) x 10(6) receptors per cell. Antagonist competition experiments displayed the order of potency: yohimbine greater than rauwolscine greater than phentolamine much greater than prazosin. Agonist competitions demonstrated the order of potency: p-aminoclonidine greater than (-)epinephrine much greater than (+)epinephrine much greater than (-)isoproterenol. This pharmacological profile is characteristic of the human platelet alpha 2-adrenergic receptor. The expressed receptor is able to couple to the Gi protein. Thus, when epinephrine competition for specific binding of [3H]rauwolscine was performed in the presence of 1 mM MgCl2, 1 mM Gpp[NH]p increased the Ki for epinephrine from 164 to 315 nM. Following preincubation of cultures with 1 mM isobutylmethylxanthine, 1 microM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 microM epinephrine. A transfected clone which did not demonstrate detectable alpha 2-adrenergic receptor mRNA displayed low levels of alpha 2-adrenergic receptor, (less than 50 fmol/mg membrane protein), similar to those found in the parent C127 cell line. In this clone, epinephrine did not attenuate but, rather, enhanced forskolin-stimulated cyclic AMP accumulation. This new C127 cell line expressing high levels of alpha 2-adrenergic receptor provides an abundant source of a single human adrenergic receptor subtype in membrane-bound conformation which is able to couple to the Gi protein and inhibit forskolin-stimulated adenylate cyclase activity. This cell line will facilitate studies of the structure: function relationship of the alpha 2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor.     

Alpha 1 adrenergic receptor control of renal blood vessels during aging

Aging humans and rats have a reduced renal vascular constriction response to stress, change in posture, or exercise. In this study, renal interlobar arteries from 9- (intermediate age) to 15-month-old (aging) male Wistar rats constricted less to alpha-adrenergic agonists than those of 4-month-old (young adult) rats. The reduced contraction to A61603 (alpha 1 A agonist) was similar to that to norepinephrine and phenylephrine. Therefore, it appears that the reduction in constriction is primarily related to alpha 1 A receptor stimulation. GeneChip microarray hybridization analysis of the interlobar arteries with the RAE 230A GeneChip indicated that there were no significant differences in gene expression for alpha 1 A/C, 1B, or 1D receptors between 4-month-old (young adult) and 1-year-old (aging) male Wistar rats. Competitive binding experiments (prazosin) revealed that maximal binding (Bmax, fmol/mg protein) of the alpha 1 receptors of interlobar arteries was reduced 25% by 10 months of age and 50% by 18+ months of age. Alpha 1 receptor-induced arterial constriction and prazosin binding were both down-regulated. The loss of receptor-initiated constriction likely includes down-regulation of maximum agonist binding by alpha 1 adrenergic receptors.     

Heterogeneity of alpha 1 receptors associated with vascular smooth muscle: evidence from functional and ligand binding studies

The nature of the alpha 1 receptor associated with rabbit aorta has been examined in functional and receptor binding studies. In isolated aortic rings the dose-response curve for (-)metaraminol was not parallel to that of (-)epinephrine, (-)norepinephrine or (-)phenylephrine. Following inactivation of a portion of the alpha receptors with phenoxybenzamine, the occupancy versus response relationship for metaraminol, in contrast to the other test agonists, was biphasic. These results suggest the possibility that metaraminol interacts with different functional groups on the alpha 1 receptor than the other test agonists. In microsomes prepared from frozen aorta, metaraminol bound to two classes of sites (KH = 0.41 +/- 0.12 microM, KL = 39.1 +/- 7.1 microM) labelled by the selective alpha 1 antagonist [3H] prazosin. Similar binding characteristics were observed in microsomes prepared from aorta shipped in serum on ice or aorta from animals killed in our laboratory. Norepinephrine also bound to two sites on the alpha receptor in all three preparations tested (KH = 0.06 +/- 0.01 microM, KL = 5.09 +/- 2.4 microM; estimates from frozen aorta). The Scatchard plot of [3H]prazosin binding to microsomes prepared from frozen aorta was curvilinear. Estimates of the affinities and site densities were 49.6 +/- 15.3 pM and 44.8 +/- 11.8 pmol/gm protein and 1.0 +/- 0.2 and 43.8 +/- 17.4 pmol/gm for the high and low affinity sites, respectively. These data are consistent with the idea that there are subtypes of the alpha 1 receptor.