Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

Characterization of Rat Cerebral Cortical Beta Adrenoceptor Subtypes Using (-)-[125I]-Iodocyanopindolol

Abstract

(-)-[¹²⁵I]-Iodocyanopindolol ((-)ICYP), used to characterize beta adrenoceptors on membrane preparations from rat cerebral cortex, was shown to have affinity for both beta adrenoceptors and serotonin receptors. Therefore, 10 µM serotonin was added to the assays to prevent (-)ICYP binding to serotonin receptors. Under these conditions, (-)ICYP binding to the cortical membrane preparation was reversible and saturable, and the association reaction was very slow. The dissociation reaction was also very slow, and revealed two affinity states corresponding to a high and a low affinity state. Scatchard analysis showed a single class of binding sites with an equilibrium dissociation constant (K_D) of 20.7 pM, and a maximal density of binding sites (B_max) of 95.1 fmol/mg membrane protein. Displacement binding analyses revealed a potency series of (-) isoproterenol greater than (-) epinephrine equal to (-) norepinephrine, suggesting a predominance of the beta₁ adrenoceptor subtype. Detailed competition ligand binding studies with the selective beta₁ adrenoceptor antagonist ICI-89406 and the selective beta₂ adrenoceptor antagonist ICI-118551, showed that about 70% of the beta adrenoceptor population in the rat cortex is of the beta₁ subtype with the remainder being of the beta₂ subtype.

We conclude that since (-)ICYP binds to both beta adrenoceptors and serotonin receptors, it is important to prevent the binding of (-)ICYP to serotonin receptors by adding a suppressing ligand like excess cold serotonin when assaying beta adrenoceptors. We have presented the first such characterization of rat cerebral cortical beta adrenoceptors with (-)ICYP in this study.     

Post-natal evolution of rat cardiac beta-adrenoceptors

Cardiac beta-adrenoceptors (beta AR) were studied using membranes prepared at birth (day 0) and at days 7, 10, 15, 21, 30, 45 and 60. Saturation experiments using the antagonist ligand (125I)-iodocyanopindolol (ICYP) allowed the determination of beta AR number (Bmax) and ICYP dissociation constant (Kd), while (-)isoproterenol competition curves of ICYP binding, performed in the absence or presence of Gpp(NH)p (10(-4) M), were used to measure the relative proportions of high and low affinity states of the beta AR for the agonist and to assess the ability of beta AR to couple with the GTP-binding protein. Rat cardiac beta AR evolved at 3 distinct periods: during the first period (days 0-10), the receptor density and ICYP Kd were half that of adults, and beta AR were present only in an homogeneous high affinity state. The second period (days 15-21) was characterized by a progressive increase in beta AR number and ICYP Kd, while analysis of (-)isoproterenol competition curves indicated that beta AR were poorly coupled to the GTP-binding protein. In the third period (days 30-60), ICYP Bmax and Kd were respectively 53.9 +/- 1.2 fmoles/mg protein and 106.4 +/- 2.9 pM, while analysis of (-)isoproterenol competition curves showed the existence of high and low affinity binding states in equal proportions in the absence of Gpp(NH)p, and of one homologous low affinity state of the receptor in its presence. These data indicate that beta AR follow a postnatal evolution marked by an increase in beta AR density concomitant with a decrease in affinity toward the antagonist ligand ICYP, accompanied by the progressive appearance of a poorly-coupled beta AR. However, the number of efficiently coupled receptors was found to be similar in adult and newborn rats.     

Identification of adenylate cyclase-coupled beta-adrenergic receptors in the developing mammalian palate

A direct radioligand binding technique utilizing a beta-adrenergic antagonist [3H]Dihydroalprenolol [( 3H]DHA) was employed in the identification and characterization of fetal palatal beta-adrenergic receptors. [3H]DHA binding was saturable (Bmax 16 fmol/mg protein) with high affinity and an apparent equilibrium dissociation constant (KD) of 1.5 nM. Binding of [3H]DHA was displaced by the competitive beta-adrenergic antagonist propranolol in a concentration-dependent manner. Dissociation kinetic studies demonstrated almost complete reversibility of radioligand binding within 60 min. The functionality of these beta-adrenergic receptors was demonstrated by showing that fetal palatal mesenchymal cells responded to catecholamine agonists with dose-dependent accumulations of intracellular cAMP. This effect could be entirely blocked by the beta-antagonist, propranolol. The relative potency order of catecholamines in eliciting an elevation of cellular cAMP was characteristic of a beta 2-adrenergic receptor-mediated response: (-) isoproterenol greater than (-) epinephrine greater than (-) norepinephrine. In addition, this response was found to be stereospecific with (-) isoproterenol being significantly more potent than (+) isoproterenol. Both the [3H]DHA binding characteristics and the catecholamine sensitivity of fetal palatal tissue support the presence of adenylate cyclase-coupled beta-adrenergic receptors in the developing mammalian secondary palate.     

Characterization of beta-adrenergic binding sites on rodent Leydig cells

A radioligand binding technique was used to study beta-adrenergic binding sites on rodent Leydig cells. Beta-Adrenergic binding sites were found on Leydig cells in both the rat and mouse. Binding of [3H]CGP-12177 [4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazole-2-one] to purified rat Leydig cells was found to be saturable, temperature and time dependent, stereospecific, and readily reversible by the beta-adrenergic antagonist propranolol. Scatchard analysis revealed the presence of high-affinity sites with an apparent dissociation constant (Kd) of 0.79 +/- 0.22 nM and maximal binding capacity (Bmax) of 1716 +/- 245 sites per rat Leydig cell. Competition of various beta-adrenergic agonists and antagonists with [3H]CGP indicates an order of potency of L-isoproterenol greater than epinephrine = salbutamol greater than norepinephrine greater than D-isoproterenol and dl-propranolol = ICI 118,551 much greater than atenolol, respectively. These observations suggest that the binding sites are predominantly of the beta 2-receptor subtype. Incubation of freshly isolated rat Leydig cells with luteinizing hormone (100 ng/ml) caused consistent stimulation of androgen production, but only occasional stimulation by the beta-agonist isoproterenol (10 microM) was observed. However, these cells consistently responded to the beta-agonist after 3 h in primary cultures. These findings indicate that rodent Leydig cells possess beta-adrenergic binding sites and point out a possible dissociation between receptor recognition and physiologic response.     

Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. [3H]Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, [3H]Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.     

Dibutyryl-cAMP increases functions of 5-hydroxytryptamine2 receptors, but not of beta 2-adrenergic receptors, in a clonal cell line of rat neurotumor RT4.

A peripheral nervous system cell line RT4-B, established by Imada and Sueoka (Dev. Biol., 66:97-108, 1978), was shown to respond to serotonin [5-hydroxytryptamine (5-HT)] and catecholamines. 5-HT induced a small and transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) in the RT4-B cells. The increase was effectively blocked by 5-HT2 receptor antagonists (spiperone, ritanserin and mianserin), but not by a 5-HT3 receptor antagonist (MDL72222), or a alpha 1-adrenergic receptor antagonist (prazosin), indicating that RT4-B cells express 5-HT2 receptors. On the other hand, catecholamines increased cyclic AMP production by RT4-B. The order of potency for stimulating cyclic AMP synthesis was isoproterenol greater than epinephrine much greater than norepinephrine much greater than dopamine, and the stimulation was effectively inhibited by the nonselective beta-adrenergic receptor antagonist propranolol, but not by the beta 1-adrenergic receptor antagonist atenolol, suggesting that RT4-B cells express beta 2-adrenergic receptors. The differentiating agent N6,2'-O-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) enhanced the 5-HT-induced [Ca2+]i increase, but not the catecholamine-induced cyclic AMP production. The increase in the 5-HT response paralleled the increase in the density of 5-HT2 receptors. n-Butyric acid (2 mM) and 8-bromoadenosine 3',5'-monophosphate (1 mM) also increased the 5-HT response, and the sum of these increases was nearly equal to that induced by dibutyryl-cAMP. These results indicate that RT4-B is a novel model cell line for the study of 5-HT2 and beta 2-adrenergic receptors and their second messenger responses and for the analysis of the mechanisms how 5-HT2 receptor gene expression is controlled.     

Characterization of Leydig cell gonadotropin-releasing hormone binding sites utilizing radiolabeled agonist and antagonist

Gonadotropin-releasing hormone (GnRH) binding sites in intact Leydig cells and in membrane preparations were investigated using 125I-labeled GnRH agonist and antagonist. Binding was saturable and involved a single class of high affinity sites. Intact Leydig cells and a membrane preparation had a higher affinity for GnRH agonist (Kd 3.0 +/- 1.7 X 10(-10) M) than for GnRH antagonist (Kd 10.0 +/- 1.8 X 10(-10) M). With anterior pituitary membranes the Kd was 2.8 +/- 0.7 X 10(-10) M for the agonist and 2.4 +/- 1.4 X 10(-10) M for the antagonist. The Kd for GnRH was similar for Leydig cells and the anterior pituitary. Chymotrypsin and trypsin digestion decreased receptor binding, but neuraminidase increased Leydig cell binding in contrast to the decrease in binding observed with pituitary receptors. The results suggest that the Leydig cell GnRH binding sites may differ from the pituitary receptor which may be related to structural differences in GnRH-like peptides recently described in extracts of rat testis.     

Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes.

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.     

Catecholamine-induced stimulation of testosterone production by Leydig cells from fetal mouse testis

In contrast to the strong stimulation of testosterone production by hCG, L-isoproterenol had little effect on freshly isolated Leydig cells from 18-day-old mouse fetuses. However, the ability of fetal Leydig cells to respond to L-isoproterenol exposure increased during culture (0-24 h). The response of the cultured cells to L-isoproterenol was dose-dependent with an ED50 at 2 X 10(-7) M. Adrenaline and noradrenaline at a concentration of 10(-5) M also increased testosterone production by cultured fetal Leydig cells. DL-Propranolol, a beta-antagonist, inhibited L-isoproterenol-stimulated testosterone production in a dose-dependent manner, while phentolamine, an alpha-adrenergic antagonist, had no effect. These results suggest that catecholamines may play an essential role in the control of testicular steroidogenesis during fetal development.     

Beta-adrenergic receptor binding characteristics and responsiveness in cultured Wistar-Kyoto rat arterial smooth muscle cells

The tone of arterial blood vessels is regulated by the catecholamines through their receptors on arterial smooth muscle cells (ASMC). beta 2-adrenergic receptors of ASMC mediate vasodilation through agonist mediated c-AMP production. Previous reports have described these receptors on freshly isolated blood vessels. This study demonstrates the presence of beta 2-adrenergic receptors on cultured rat ASMC and that these receptors are functional. beta-adrenergic receptor binding was measured using [3H]-dihydroalprenolol (DHA) binding to the membrane of cultured ASMC from normotensive Wistar-Kyoto rats. The ASMC beta-adrenergic receptors have a Kd of 0.56 +/- 0.16 nM and a Bmax of 57.2 +/- 21.7 fmol/mg protein. Competition binding studies revealed a much greater affinity of these receptors for epinephrine than norepinephrine, indicating the preponderance of a beta 2-adrenergic receptor subtype. Isoproterenol stimulation of cultured ASMC resulted in a 14 +/- 7 fold increase in intracellular c-AMP content of these cells indicating these receptors are functional. beta-adrenergic receptors of cultured ASMC provide an excellent system in which the association between hypertension and observed beta-adrenergic receptor differences can be further explored.     

Modulation of mouse Leydig cell steroidogenesis through a specific arginine-vasopressin receptor

Characterization of specific vasopressin binding sites was investigated in purified mouse Leydig cells using tritiated arginine-vasopressin. Binding of radioligand was saturable, time- and temperature-dependent and reversible. (3H)-AVP was found to bind to a single class of sites with high affinity (Kd = 2.20 +/- 0.18 nM) and low capacity (Bmax = 17.4 +/- 1.8 fmol/10(6) Leydig cells). Binding displacements with specific selective analogs of AVP indicated the presence of V1 subtype receptors on Leydig cells. The ability of AVP to displace (3H)-AVP binding was greater than LVP and oxytocin. The unrelated peptides, somatostatin and substance P, were less potent, while neurotensin and LHRH did not displace (3H)-AVP binding. The time-course effects of AVP-pretreatment on basal and hCG-stimulated testosterone and cAMP accumulations were studied in primary culture of Leydig cells. Basal testosterone accumulation was significantly increased by a 24 h AVP-pretreatment of Leydig cells (P less than 0.001). This effect was potentiated by the phosphodiesterase inhibitor (MIX) and was concomitantly accompanied by a slight but significant increase in cAMP accumulation (P less than 0.01). AVP-pretreatment of the cells for 72 h had no effect on basal testosterone accumulation, but exerted a marked inhibitory effect on the hCG-stimulated testosterone accumulation (P less than 0.001). This reduction of testosterone accumulation occurred even in the presence of MIX and was not accompanied by any significant change of cAMP levels. We conclude from these data that AVP is capable of modulating steroidogenesis in Leydig cells through specific and functionally V1 receptor subtype and postulate that this effect may be part of an intratesticular paracrine/autocrine control mechanism.     

Interactions of agonists and antagonists with beta-adrenergic receptors on intact L6 muscle cells

A binding assay has been developed to characterize beta-adrenergic receptors on intact L6 muscle cells. The affinity of beta-adrenergic receptors for the radioligand iodohydroxybenzylpindolol (IHYP) was the same in membrane preparations and in intact cells when determined by either equilibrium binding or kinetic analysis. The number of specific IHYP binding sites per cell was approximately the same on intact cells as on membranes. The pharmacological properties of antagonists indicated that the receptors on intact cells were identical to those on membranes. However, the beta-adrenergic receptors on intact cells had a 100-400 fold lower affinity at equilibrium for the agonist isoproterenol than did beta-adrenergic receptors on membranes. This low affinity of the receptor for agonists as measured by inhibition of radioligand binding in intact cells has also been observed in C6 (2) and S49 (3) cells. Our results suggest that beta receptors on intact cells after a 1 minute incubation was similar to the KD value for isoproterenol measured in membranes at equilibrium in the presence of GTP. After 1-2 minutes of exposure to a low concentration of agonist, binding of IHYP was no longer inhibited. These results suggest that agonists rapidly convert the beta receptors on intact cells to a state which has a low affinity for agonists. The affinity of the receptor for antagonists did not change during the incubation.     

Regulation of 1,4-dihydropyridine and beta-adrenergic receptor sites in coronary artery smooth muscle membranes.

The receptor sites for 1,4-dihydropyridine (DHP) calcium channel ligands were identified and pharmacologically characterized in partially purified canine coronary artery smooth muscle (CSM) membranes (purification factor for 1,4-DHPs 2.8 and 2.2 respectively) using Ca2+ channel agonist (-)-S-[3H]BAYK 8644 and antagonist (+)-[3H]PN 200-110 as radioligands. The beta-adrenergic receptors were identified with (-)-3-[125I]iodocyanopindolol (ICYP). Specific binding of 1,4-DHPs and ICYP to membrane fraction was saturable, reversible and of both high and low affinity. The Kd for 1,4-DHP Ca2+ channel agonist was 0.59 +/- 0.05 and for antagonist 0.35 +/- 0.06 nmol/l and for low affinity binding sites Kd = 9.0 +/- 0.18 and 18.0 +/- 1.1 nmol/l. The high affinity 1,4-DHP binding (Bmax = 265 +/- 21 and 492 +/- 12 fmol/mg protein), showed stereoselectivity, temperature-dependence as well as pharmacological specificity: isoprenaline- and GTP-sensitivity, positive modulation with dilthiazem and negative modulation with verapamil, that is, properties characteristic of 1,4-DHP receptor sites on L-type Ca2+ channels. The low affinity binding sites were characterized as nonselective, temperature independent, dipyridamol-sensitive and represented a nucleoside transporter. The proportion of high affinity binding sites identified in the CSM membranes was 1.85 : 1.0 in favour of the antagonist. Results obtained with [125I]omega Conotoxin GVI A demonstrated that CSM membrane fractions isolated from median layers of coronary artery were devoid of substantial contamination with fragments of neuronal cells.     

Non-Specific Binding of the Fluorescent B-Adrenergic Receptor Probe Alprenolol-NBD

Abstract

The fluorescent ß-adrenergic receptor probe alprenolol-NBD was found to exhibit a high affinity (K_d 3.2 nM) and a low capacity (10 fmol/mg protein) for the ß₂-adrenergic receptor on living Chang liver cells but also a high affinity (K_d 320 nM) for non-ß-adrenergic receptor binding sites with a very high capacity (28,000 fmol/mg protein). Calculations are presented which make clear that less than 3% of the binding of alprenolol-NBD during visualization experiments is ß-adrenergic receptor related. Furthermore, it is shown that besides the downregulation of ß-adrenergic receptors during incubation with isoproterenol, the high-affinity non-ß-receptor binding sites are also deminishing during incubation with isoproterenol. Based on our findings it is concluded that the results of Henis et al. (1) who claimed the visualization of the ß-adrenergic receptor population on Chang liver cells by alprenolol-NBD must be interpreted as an almost completely non-specific fluorescence.     

The (--)[3H]dihydroalprenolol binding to rat adipocyte membranes: an explanation of curvilinear Scatchard plots and implications for quantitation of beta-adrenergic sites

In rat adipocyte membranes, both beta-adrenergic agonists and beta-adrenergic antagonists competed with (--)[3H]dihydroalprenolol for high affinity (KD 2-4 nM) and low capacity binding sites. The antagonists but not the agonists competed with (--)[3H]dihydroalprenolol for lower affinity and higher capacity sites. The present studies were performed in order to characterize the adipocyte beta-adrenergic receptor and distinguish it from low affinity, higher capacity sites which were heat-labile and not stereoselective. When isoproterenol was used to define the nonspecific binding, saturation studies showed a single binding site with a capacity of approximately 100 fmol/mg membrane protein (corresponding to approximately 50,000 sites/adipocyte). Binding was saturated by 10 nM (--)[3H]dihydroalprenolol. Approximate KD's of 204 nM were observed. Kinetic analysis of (--)[3H]dihydroalprenolol binding provided an independent measurement of KD between 0.75 and 1.1 nM. This binding site had the characteristics of a beta 1-adrenergic receptor with the potency of isoproterenol greater than norepinephrine greater than or equal to epinephrine as competitors of binding. Furthermore, the KD of inhibition of (--)[3H]dihydroalprenolol binding correlated with the Ki of inhibition by antagonists or Ka of activation by agonists of glycerol release in isolated adipocytes (r = 0.968, P less than 0.001). These results suggest that beta-adrenergic agonists compete with (--)[3H]dihydroalprenolol for the high affinity binding site which represents the physiological site. Furthermore, the use of antagonists (propranolol, alprenolol) to define specific beta-binding includes nonspecific site(s) as well as the beta-adrenergic site. Previous characterization and quantitation of beta receptors in rat fat cell membranes may have been in error by incorporating both types of binding in their measurement.     

Cross-regulation between G-protein-coupled receptors. Activation of beta 2-adrenergic receptors increases alpha 1-adrenergic receptor mRNA levels.

Stimulation of DDT1 MF-2 vas deferens cells with epinephrine resulted in a time- and dose-dependent loss of alpha 1-adrenergic receptor-specific ligand binding. Regulation of alpha 1-adrenergic receptor mRNA was characterized. In monolayer culture, cells displayed 0.7 +/- 0.05 amol of alpha 1-adrenergic receptor mRNA/microgram of total cellular RNA. Epinephrine, which acts at both alpha 1- and beta 2-adrenergic receptors of DDT1 MF-2 cells, induced a short term (2-8 h) increase (50-70%) in the abundance of alpha 1-adrenergic receptor mRNA. Propranolol, a beta 2-adrenergic receptor antagonist, attenuated the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA but did not affect the decrease in alpha 1-adrenergic receptor-specific ligand binding. Phentolamine, an alpha 1-adrenergic receptor antagonist, did not attenuate the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA at 4 h but did block the decrease in alpha 1-adrenergic receptor-specific ligand binding. The half-life of the alpha 1-adrenergic receptor mRNA was approximately 7 h in untreated cells as well as in cells challenged with epinephrine. The epinephrine-promoted increase in alpha 1-adrenergic receptor mRNA was found to result from cross-regulation via beta 2-adrenergic receptors. Cholera toxin, forskolin, as well as the cyclic AMP analog CPT cAMP (8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate) increased the alpha 1-adrenergic receptor mRNA at 4 h, as did epinephrine in the presence of alpha 1-antagonists but not in the presence of a beta-adrenergic antagonist. This is the first report of heterologous up-regulation of mRNA levels of adrenergic receptors. Cross-regulation between alpha 1- and beta 2-adrenergic receptor-mediated pathways at 4 h occurs at the level of mRNA whereas later down-regulation of alpha 1-receptor mRNA and binding proceed via agonist activation of alpha 1-adrenergic receptors.     

Characterization of monoclonal antibodies to the beta-adrenergic antagonist alprenolol as models of the receptor binding site

Antibodies to receptor ligands have been valuable in understanding the nature of receptor-ligand interactions. We have developed four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol by immunizing A/J mice with (-)-alprenolol coupled to keyhole limpet hemocyanin. The antisera from these mice displayed specific [3H]dihydroalprenolol ([3H]DHA) binding that was inhibited by alprenolol, propranolol, and isoproterenol. Somatic cell fusion of spleen cells from the immunized mice to SP2/0 myeloma cells, followed by limited dilution subcloning, resulted in the isolation of four hybridomas (1B7, 5B7, 5D9, and 2G9) demonstrating three different classes of ligand binding characteristics. 1B7 had the highest binding affinity for antagonists based on Scatchard analysis (Kd [125I]- CYP = 1.4 X 10(-10) M; Kd [3H]DHA = 6.5 X 10(-9) M), and was the only antibody to demonstrate agonist-inhibition of [3H]DHA binding. Ki values computed from competitive inhibition curves of [3H]DHA binding to 1B7 resulted in a rank order of potency similar to that of beta-2-adrenergic receptors: (-)-propranolol greater than acebutolol amine greater than isoproterenol greater than (+)-propranolol greater than epinephrine greater than norepinephrine. 5B7 and 5D9 exemplified a second class of antibody. This pair had lower antagonist binding affinities (Kd [3H]DHA = 2 X 10(-8) M and 2.5 X 10(-7) M, respectively) and was stereoselective in binding receptor antagonists: (-)-propranolol greater than (+)-propranolol greater than acebutolol amine. Agonist inhibition of [3H]DHA binding to these antibodies could only be observed at very high concentrations (greater than 10(-4) M agonist), and was not dose-dependent. Finally, the class of anti-alprenolol monoclonal antibodies represented by 2G9 had the lowest antagonist binding affinity of all (IC50 alprenolol = 1 X 10(-5) M), did not demonstrate ligand stereoselectivity, and did not recognize agonists. We propose that antibodies raised against beta-adrenergic receptor ligands demonstrating stereoselective agonist binding will also demonstrate high affinity antagonist binding, and that they will closely parallel the binding characteristics of the receptor. According to this "agonist best-fit hypothesis," anti-idiotypic antibodies raised against the binding site of these idiotypes might contain true mirror images of the beta-adrenergic receptor binding site.     

Evidence for a Leydig cell progesterone receptor in the rat

Tritiated promegestone [3H] R 5020 is bound with high affinity by charcoal-treated cytosol prepared from purified Leydig cells. The binding is characterized by high affinity (Kd = 2 x 10(-9) M) and specificity (R 5020 = progesterone greater than testosterone = dehydrotestosterone greater than hydroxyprogesterone greater than cortisol = dexamethasone greater than estradiol) appropriate for progesterone receptors. In vitro, progestin-bound cytosol was quantitatively translocated to nuclei fractions, only if cytosol samples were previously labeled at 25 degrees C. However no translocation of binding activity was observed when previous cytosol labeling was done in the presence of sodium molybdate. Effects of glucocorticoids, androgens and estrogens on the Leydig cell are well documented, the demonstration of a putative progesterone receptor raises the possibility of direct effect of progesterone on the Leydig cell.