Structural and functional roles of small group-conserved amino acids present on helix-H7 in the ß(2)-adrenergic receptor.

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix-helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2-H4 in the ß(2)-adrenergic receptor (ß(2)-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315(7.42) and Ser319(7.46), on H7 for structure-function analysis. Replacing Ser319(7.46) with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15-20% agonist-independent activity. Replacement of Ser319(7.46) with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315(7.42) and Ser319(7.46) are stabilizing ß(2)-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315(7.42) with Trp286(6.48) and hydrogen bonding interactions of Ser319(7.46) with amino acids on H1-H2-H7 and with structural water.

Non-steroidal anti-inflammatory drugs inhibit epinephrine- and cAMP-mediated lipolysis in isolated rat adipocytes.

Acute ethanol intoxication increased triacylglycerides (TAG) and thiobarbituric acid reactive substances (TBARS) in liver and promoted the liberation of epinephrine. Four non-steroidal anti-inflammatory drugs (NSAIDs)--aspirin, naproxen, nimesulide and piroxicam--prevented this increase in TAG and TBARS. Because fatty acids provided by adipose tissue contribute substantially to elevated hepatic TAG in ethanol-intoxicated rats, it was thought that the NSAIDs might reduce epinephrine-stimulated lipolysis in these rats. Isolated rat adipocytes were activated with epinephrine in the presence or absence of the NSAIDs. The NSAIDs inhibited epinephrine-stimulated lipolysis. These drugs did not modify the binding of dihydroalprenolol (beta-adrenergic agonist) to their receptors in isolated guinea-pig liver membranes. The NSAIDs, at concentrations 3,000-fold lower than that of cAMP, inhibited stimulated lipolysis by this messenger. In conclusion, aspirin, naproxen, nimesulide and piroxicam reduce the release of fatty acids from adipose tissue to the liver by inhibiting the epinephrine-stimulated lipolysis, and this, in part, explains the protective action of these NSAIDs against hepatic signs of acute ethanol intoxication.

Rapid electrical stimulation of contraction reduces the density of beta-adrenergic receptors and responsiveness of cultured neonatal rat cardiomyocytes. Possible involvement of microtubule disassembly secondary to mechanical stress.

BACKGROUND: Although tachycardia is commonly present in patients with congestive heart failure, its role in the development of congestive heart failure remains unclear. We studied the effect of rapid electrical stimulation of contraction on beta-adrenergic receptor (beta-AR) signal pathway in cultured cardiomyocytes of neonatal rats. METHODS AND RESULTS: Contraction of cardiomyocytes was induced by electrical stimulation at 50 V with twice the threshold pulse width. beta-ARs were identified by [(3)H]CGP-12177 and [(3)H]dihydroalprenolol. Electrical stimulation reduced cell-surface but not total beta-AR density; the effect was dependent on pacing frequency (a reduction of 11%, 28%, and 18% in cells paced at 2.5, 3. 0, and 3.3 Hz, respectively). This reduction was apparent at 3 hours, in contrast to reduced beta-AR density after exposure to isoproterenol (ISP) for 1 hour. The fraction and inhibition constant of beta-AR binding agonist with high affinity were not affected by rapid electrical stimulation. In cardiomyocytes paced at 3.0 Hz for 24 hours, the response to ISP decreased compared with unpaced cells, 142% versus 204% of baseline with 1 micromol/L ISP, whereas the responses to forskolin or acetylcholine were not different. Treatment of cardiomyocytes with 2,3-butanedione monoxime (10 mmol/L) or taxol (10 micromol/L) inhibited the rapid pacing-induced reduction in beta-AR density. CONCLUSIONS: Our results suggest that contractile activity is involved in regulation of cardiac function by modulating the beta-AR system independently of hemodynamic and neurohormonal factors. This may help to elucidate the role of mechanical stress in the development of heart failure.

Expression of beta-adrenergic receptors in the rat uterus: effects of puberty and oestrogen treatment during prepubertal development.

The expression of beta-adrenoceptors in the rat uterus has been analysed during the peripubertal transition and following acute and chronic oestradiol treatment during prepubertal development. The distribution and density of beta-adrenoceptors was assessed autoradiographically on cryostat tissue sections using [3H]-dihydroalprenolol ([3H]-DHA). Binding sites were localised in all ages and experimental situations examined and showed the following intensity of labelling: endometrial epithelium > longitudinal muscle layer > circular myometrial layer > endometrial stroma. Competition experiments with the selective antagonists ICI 118,551 and atenolol, showed that most of the beta-adrenoceptors in the uterus belong to the beta(2) receptor subclass. In prepubertal animals, the density of [3H]-DHA binding sites was extremely low. Following puberty the density of binding sites showed a generalised increase. Acute administration of oestradiol at the end of the prepubertal period provoked an increase in the density of [3H]-DHA binding sites in all uterine regions, but the levels of labelling were lower than in peripubertal animals at proestrus and oestrus. Following chronic oestrogen treatment during postnatal development, oestradiol increased further the density of [3H]-DHA binding sites. Results are discussed considering both the endocrine and neural changes accompanying puberty and oestradiol treatment.

Tannin inhibits the cAMP-beta-adrenergic receptor pathway in bovine tracheal epithelium.

Tannin, isolated from cotton bracts, inhibits chloride secretion in airway epithelium. In bovine tracheal epithelial cells, tannin (25 micrograms/ml) blunted isoproterenol (Iso)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Inhibition was time and dose dependent, with 52 +/- 5% (mean +/- SE, n = 6) inhibition at 60 min and 82 +/- 9% (n = 3) inhibition at 8 h. Inhibition was reversible starting at 4 h. Low-molecular-mass tannin (1,000-5,000 Da) had no effect on Iso-stimulated cAMP accumulation, whereas N-acetylcysteine, which interacts with cysteine residues, blocked the effects of tannin on Iso-stimulated cAMP accumulation. Tannin exposure (25 micrograms/ml for 30 min) had no effect on the dissociation constant (Kd) for [3H]dihydroalprenolol (DHA) (0.41 +/- 0.03 nM, n = 3) but decreased maximal binding from 252 +/- 32 to 162 +/- 36 fmol/mg protein. Using single-point analysis and [3H]CGP-12177, we determined that tannin (25 micrograms/ml for 4 h) decreased surface beta-adrenergic receptor density from 26.4 +/- 4.3 (n = 12) to 11.9 +/- 3.0 fmol/mg protein and that the decrease was dose dependent. Agonist binding affinity by Iso displacement of DHA demonstrated a two-site model (Kd values = 27 +/- 9 and 2,700 +/- 600 nM) and a ratio of high- to low-affinity receptors of 1:1. Tannin (25 micrograms/ml) steepened the curve and shifted it to the right, as did Gpp(NH)p. Gpp(NH)p had no further effect on the shape or position of the displacement curve in the presence of tannin. In contrast, when polymer length was decreased by oxidation, tannin had no effect on the DHA displacement curve. These data demonstrate that tannin reversibly desensitizes bovine tracheal epithelial cells to Iso, decreases beta-adrenergic receptor density, and uncouples the receptor from its stimulatory G protein. These data also suggest that the polymer length of tannin and its interaction with cysteine residues are important for these effects. These studies provide additional evidence for the role of tannin in the occupational lung disease byssinosis.

Structure-affinity relationships and stereospecificity of several homologous series of local anesthetics for the beta2-adrenergic receptor.

UNLABELLED: Local anesthetics inhibit binding of ligands to beta2-adrenergic receptors (beta2ARs), and, as a consequence, inhibit intracellular cAMP production. We hypothesized that among homologous local anesthetics, their avidity at inhibiting binding of tritiated dihydroalprenolol (3H-DHA) to beta2ARs would increase with increasing length of alkyl substituents and would demonstrate stereospecificity. Specific binding of 3H-DHA to human beta2ARs was assayed in the presence of six different members of the 1-alkyl-2,6-pipecoloxylidide class of local anesthetics (including mepivacaine, ropivacaine, and bupivacaine), the R(+) and S(-) bupivacaine enantiomers, lidocaine, prilocaine, etidocaine, procaine, and tetracaine. Avidity of binding to beta2ARs increased with increasing length of the alkyl chain (pKi values = 2.4, 3.6, 4.3, 4.1, 4.1, 5.9 for the methyl [mepivacaine], ethyl, S(-)propyl [ropivacaine], butyl [bupivacaine], pentyl, and octyl derivatives, respectively). We found no evidence for bupivacaine stereospecificity (pKi values = 4.3 and 4.9 for the S(-) and R(+) isomers, respectively). Other amide and ester local anesthetics also showed increasing potency with increasing length of alkyl substituents (pKi values = 3.6, 3.8, and 4.3 for lidocaine, prilocaine, and etidocaine; 4.2 and 5.6 for procaine and tetracaine, respectively). The correlation between increased inhibition of beta2AR binding and alkyl chain length resembles the correlation between local anesthetic potency at nerve block and increased alkyl chain length. The lack of clear stereospecificity is consistent with the relatively low potency these agents demonstrate at inhibition of beta2AR binding. Finally, the relatively potent inhibition of beta2ARs by etidocaine, tetracaine, and bupivacaine suggests that their propensity for cardiovascular depression after accidental intravenous overdose could result from beta2AR or beta1AR blockade and inhibition of cAMP production. IMPLICATIONS: Local anesthetics demonstrate a rank order of avidity for displacing ligands from beta2-adrenergic receptors such that larger molecules displace ligands at lower concentrations than smaller local anesthetic molecules. This relationship between molecular size and receptor avidity could explain the greater propensity for cardiovascular toxicity of relatively large local anesthetics such as bupivacaine.

Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.

Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.

Antibodies to beta 1 and beta 2 adrenoreceptors in Chagas' disease.

Evidence accumulated over the last decade concerning human and experimental models suggests that an immunopathological mechanism may be involved in the pathogenesis of chronic Chagas' disease. In this paper we demonstrate the existence of two different circulating IgG in chagasic patients which bind with myocardial beta 1 and spleen cell beta 2 adrenoceptors, acting as non-competitive inhibitors. Both chagasic IgG against beta 1 and beta 2 adrenoceptor increased intracellular levels of cAMP that could be blocked by specific beta 1 and beta 2 adrenoceptor antagonists. The specificity for beta 1 and beta 2 adrenoceptors and the independence of other tissue reactive antibodies was demonstrated by IgG absorption with turkey red blood cell (TRBC), human lymphocytes (HL) or guinea pig red blood cells (GPRBC). The F(ab')2 fraction acted similarly. This supports the specificity of beta 1 and beta 2 adrenoceptors to the chagasic IgG and the independence of the other tissue reactive antibodies, such as EVI system. The probable pathogenic role of both beta 1 and beta 2 adrenergic chagasic antibody is discussed.

Effects of beta-2 agonist on metabolic regulation in the fetal lamb lung.

To study the effects of beta-2 agonist on metabolic regulation in fetal lamb lung, ritodrine hydrochloride, a preferential beta-2 agonist, was infused i.v. at a rate of 1.3 +/- 0.4 micrograms/kg/min (mean +/- S.D.) for 24 hr into six twin chronically catheterized fetal lambs starting between 0.86 and 0.91 gestation. Lung glycogen was depleted 56% in the ritodrine-infused twins and glycogen phosphorylase a activity was increased 1.8-fold whereas glycogen synthase activity remained unchanged. Cyclic AMP-dependent protein kinase activity increased 1.7-fold, calcium-calmodulin-dependent protein kinase (phosphorylase kinase) activity increased 1.4-fold and calcium-phospholipid-dependent protein kinase (protein kinase C) activity increased 1.6-fold. In addition, the maximal binding capacity of pulmonary beta receptors decreased 49% in the ritodrine-infused twins. However, lung cyclic AMP content was unchanged after 24 hr of ritodrine infusion. We conclude that beta-2 agonist activates protein kinases, depletes glycogen and reduces the binding capacity of beta receptors in the fetal lamb lung. We speculate that these adrenergic mechanisms are involved in regulating the effects of beta-2 agonist on fetal lung liquid and surfactant production.

[Characteristics of beta-adrenoreceptors of L-1210 leukemia and its sarcolysine-resistant variant]. / Kharakteristika beta-adrenoretseptorov kletok leikoza L-1210 i ego varianta, ustoichivogo k sarkolizinu.

Beta-adrenoceptors have been discovered on the surface of cells of leukaemia L1210 and its variant resistant to sarcolysine (D, L-melphalan). One type of functionally active receptors with dissociation constant Kd for L-[3H] dihydroalprenolol about 0.02 nM and 360 receptors per cell have been revealed in leukaemia L1210 cells. In the resistant cells two types of functionally inactive receptors with Kd1 approximately 0.02 nM (420 receptors per cell) and Kd2 approximately 2.5 nM (3000 receptors per cell) have been revealed. This property of beta-adrenoceptors may be one of the causes of tumour cell resistance to sarcolysine.

Acute change in the cyclic AMP content of rat mammary acini in vitro. Influence of physiological and pharmacological agents.

The cyclic AMP content of acini, freshly prepared from mammary tissue of lactating rats, was measured during incubation in vitro. Neither adrenergic agonists nor cyclic AMP phosphodiesterase inhibitors alone caused a change of more than 2-fold in the basal cyclic AMP content of acini. Together, however, these agents provoked increases of around 20-fold in acini cyclic AMP content. Forskolin caused similar effects. The relative potency of adrenergic agonists in increasing cyclic AMP in acini, together with the ability of selective antagonists to oppose such rises, indicated that beta 2-adrenergic receptors were involved in mediating the effects. Receptor-binding experiments using [3H]dihydroalprenolol and selective beta-antagonists confirmed the predominant presence of beta 2-adrenergic receptors on acini membranes and on membranes prepared from purified mammary secretory epithelial cells. These results elucidate some previous findings [Robson, Clegg & Zammit (1984) Biochem. J. 217, 743-749; Williamson, Munday, Jones, Roberts & Ramsey (1983) Adv. Enzyme Regul. 21, 135-145], questioning the role of cyclic AMP in the regulation of lipogenesis in mammary acini.

Prenatal nicotine exposure increases adrenergic receptor binding in the rat cerebral cortex.

Adult female rats were given nicotine in drinking water for a minimum of 6 weeks prior to mating. The nicotine intake was then maintained at a level of 6.0 +/- 0.2 mg/kg/day throughout pregnancy. At birth the offspring were fostered to control females. Brain noradrenaline levels were elevated in cortex and hypothalamus in infancy but were within the control ranges in adult offspring. However, adrenergic receptor binding was significantly increased in cerebral cortex of adult offspring consistent with the view that prenatal nicotine exposure produces permanent changes in the functioning of central noradrenergic neurons. As in previous studies, the changes were found only in male offspring.

[Desensitization of beta-adrenoreceptors and its influence on manifestations of the antiradiation effect of isoproterenol]. / Desensibilizatsiia beta-adrenoretseptorov i ee vliianie na proiavlenie protivoluchevogo éffekta izoproterenola.

A study was made of the function of beta-adrenoreceptors and Chinese hamster fibroblast cAMP during their desensitization to isoproterenol. Desensitization of adenylate cyclase was demonstrated to lead to the reduction of both the cAMP response to isoproterenol and isoproterenol capacity to protect the cells from ionizing radiation. This did not entail any changes in the number of beta-adrenoreceptors or in the dissociation constant of the beta-antagonist. It is assumed that desensitization provokes functional dissociation of the beta-receptor and adenylate cyclase. Intact adenylate cyclase is an absolute must for realization of the antiradiation potency of isoproterenol.

Characterization of a beta-adrenergic receptor in porcine trachealis muscle.

To establish a model of airway smooth muscle function we studied binding of [3H]dihydroalprenolol [( 3H]DHA), a beta-adrenergic antagonist, to membrane preparations of porcine trachealis muscle and investigated the response of adenylate cyclase to l-isoproterenol in tissue and plasma membranes. [3H]DHA binding was of high affinity (Kd = 1.0 +/- 0.1 nM), was saturable (Bmax = 87.6 +/- 13.2 fmol/mg protein), and was 90% beta 2 and 10% beta 1. Adenylate cyclase activity in the membrane preparation was (in pmol.10 min-1.mg protein-1 +/- SE): basal 420 +/- 74, guanosine 5'-triphosphate (GTP) (10 micron) 600 +/- 45, GTP (10 microM) + l-isoproterenol (100 microM) 660 +/- 63, NaF (10 mM) 1,500 +/- 134, and forskolin (100 microM) 3,000 +/- 410. Guanosine 5'-diphosphate (GDP) and GTP were active cofactors; l-isoproterenol appeared to function as an effector exchanging GTP for GDP on the guanine nucleotide regulatory protein. There was close agreement of the effective dose (ED50) of the l-isoproterenol-induced relaxation (0.95 +/- 0.45 microM) and the inhibitory constant of l-isoproterenol binding (0.39 +/- 0.10 microM). l-Isoproterenol (100 microM) induced a 100% increase in adenosine 3',5'-cyclic monophosphate (cAMP) levels in tissue strips over basal activity. Investigation of the difference in adenylate cyclase activity between tissue and plasma membranes revealed that l-isoproterenol responsive adenylate cyclase was diminished after initial homogenization. Electron microscopy demonstrated disruption of all cells at this early stage of preparation. The decrease in l-isoproterenol responsive adenylate cyclase following cell rupture is different from other tissues and suggests a difference in the actions of beta-agonist in smooth muscle compared with other tissues.

Thyroid hormone regulation of beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in foetal heart.

The effect of thyroid hormone on the beta-adrenergic receptor in foetal cardiac membranes was analysed by measuring the binding of (-)[3H]DHA. The specific activities (per mg protein) of beta-adrenergic receptors decreased with advancing gestational age, whereas the total activities (per heart) increased under the similar conditions. The change in the binding affinities was not statistically significant. 1; 3,5,3'-L-triiodothyronine (T3) stimulated the (-)[3H]DHA binding capacities of the cardiac membranes of foetuses of all age groups. The enhancement in the receptor activity was completely inhibited actinomycin D or cycloheximide. The contents of epinephrine, norepinephrine and cAMP increased with advancing gestational age; but T3 had no significant effect on the catecholamines or cAMP. Similarly, the activities of the basal, NaF stimulated and Gpp(NH)p stimulated adenylate cyclase remained unaltered by T3, but the activities increased progressively with foetal maturity. The absolute values of catecholamine stimulated adenylate cyclase activities in the hearts of T3 treated foetuses were, however, higher compared to those in the untreated foetuses. The enhancement of the activities were totally blocked by the action of actinomycin D, cycloheximide or propranolol. Our results indicate that thyroid hormone enhances the number of beta-adrenergic receptor binding sites by synthesizing new receptor proteins resulting in increased catecholamine sensitivity.

beta-Adrenergic receptors in pediatric tumors: uncoupled beta 1-adrenergic receptor in Ewing's sarcoma.

beta-Adrenergic receptors were demonstrated in membrane preparations from 6 human Ewing's sarcomas and compared to those from 46 other pediatric cancers with the use of the beta-adrenergic antagonist (-)-(3H)dihydroalprenolol [(-)[3H]DHA]. In contrast to the high numbers of receptor sites found in Ewing's sarcomas (55-640 fmol x mg-1 protein; dissociation constant Kd, 1-2 nM), other childhood cancers (neuroblastoma, rhabdomyosarcoma, brain tumors, lymphoma, osteosarcoma, hepatoblastoma, yolk sac, and Wilms' tumor) contained in general fewer beta-adrenergic receptor sites. Characteristics of (-)-[3H]DHA binding were therefore more fully characterized in the Ewing's tumors. Competition of (-)-[3H]DHA binding by classical catecholamine agonists, as well as by subtype selective agents metoprolol and zinterol, demonstrated the presence of a homogeneous population of beta 1-adrenergic sites in several Ewing's tumors. Adenylate cyclase activity in all Ewing's sarcomas was enhanced by GTP and NaF. However, in spite of high numbers of beta-adrenergic receptors, (-)-isoproterenol was not very effective in the activation of adenylate cyclase activity in several of the Ewing's tumors tested. Neither guanyl-5'-yl-imidophosphate nor GTP altered agonist potency for the receptor site in these catecholamine-insensitive tumors. Hill coefficients obtained from the competition experiments with (-)-isoproterenol (in the presence or absence of guanine nucleotide) were approximately 1.0. These uncoupled receptors were resistant to N-ethylmaleimide denaturation and were densensitized only 50% during culture in the presence of (-)-isoproterenol. Thus Ewing's sarcomas are relatively rich in beta-adrenergic sites, and several tumors appear to have a coupling lesion involving guanine nucleotide-dependent regulatory protein interaction with beta-adrenergic receptors and adenylate cyclase, similar in phenotype to that described in the (unc) variant of S49 mouse lymphoma.

Daily bupropion injections for 3 weeks attenuate the NE stimulation of adenylate cyclase and the number of beta-adrenergic recognition sites in rat frontal cortex.

In rats receiving daily doses (50 mg/kg i.p. twice daily) of bupropion HCI (WellbutrinR) repeated for 21 days the Bmax of the beta-adrenergic receptor recognition sites located in the frontal cortex is reduced. This decrease is not associated with a decrease of the apparent affinity of these recognition sites. However the Vmax of the cAMP (cyclic AMP) generating system stimulated by NE is reduced suggesting that similarly to other antidepressants bupropion down regulates beta-adrenergic receptors located in the frontal cortex. Bupropion neither inhibits MAO (monoamine oxidase) nor releases biogenic amines but only weakly inhibits monoamine uptake in vitro.

Low beta-adrenergic receptor concentration on human thymocytes.

Several agents are known that can elevate cyclic AMP levels in lymphoid cells, e.g. isoproterenol, PGE1 and adenosine. We have studied the cyclic AMP increasing effect of these agents on thymocytes from mouse and man and on human peripheral T lymphocytes. In contrast to mouse thymocytes and human peripheral T lymphocytes, human thymocytes appeared to be insensitive to isoproterenol, but did respond to PGE1 and adenosine. Furthermore, the density of beta-adrenergic receptors on the cells was determined by measuring the specific binding of 3H-dihydroalprenolol. A correlation was found between the receptor density on the cells and the rise in intracellular cyclic AMP induced by isoproterenol: human thymocytes appeared to have very few beta-adrenergic receptors, in contrast to thymocytes from mouse or to T lymphocytes from human blood. We conclude that the development of beta-adrenergic receptors in T cell ontogeny is different for mice and human beings. Comparison of animal models with the situation in man should be made with caution.

The effect of estrogen and progesterone on beta-adrenergic receptor activity in rabbit lung tissue.

Study of the effect of sex steroids on the development of beta-adrenergic receptors may be essential to an understanding of the mechanisms of both lung maturation and the initiation of labor. (3H)Dihydroalprenolol (DHA) was used to quantify beta-adrenergic receptor sites in mature and immature rabbit lung tissue. DHA binding was rapid, saturable, of high affinity (dissociation constant = 2 nM), and of low capacity (246 to 576 fmoles/mg of protein), and a adrenergic competitors demonstrated both stereoselectivity ([-]isomer much greater than [+]isomer) and a rank order of potency (isoproterenol much greater than norepinephrine greater than epinephrine) characteristic of the beta-adrenergic receptor. beta-Adrenergic receptors in the lung tissue of both mature and immature female New Zealand White rabbits were investigated under various sex steroid situations. Estrogen (diethylstilbestrol, 5 micrograms/day for 10 days) increased the beta-adrenergic receptor site number in immature rabbits compared to matched controls (435 versus 339 fmoles/mg of protein, p less than 0.02 by paired t test). Addition of progesterone (diethylstilbestrol, 5 micrograms/day for 10 days, plus progesterone, 5 mg/day for 3 days) returned the beta-adrenergic receptor site number to control values (321 fmoles/mg of protein, p less than 0.01). In mature rabbits, treatment with progesterone alone (10 mg/day for 4 days) caused a significant reduction in beta-adrenergic receptor site numbers compared to untreated, matched controls (357 versus 493 fmoles/mg of protein, p less than 0.05 by paired t test). in the presence of estrogen, beta-adrenergic receptor activity is enhanced in both mature and immature rabbit lung tissue. Addition of progesterone restores this activity to control values.

Receptor-associated changes of the catecholamine-sensitive adenylate cyclase in glioma cells doubly transformed with Moloney sarcoma virus.

A doubly transformed rat glioma cell line, designated C6V-1, was obtained from rat glioma C6 cells by infection with a rat-adapted variant of Moloney sarcoma virus (MSV-M-os). The C6V-1 cells show karyotypic changes in chromosome number (43) and structure, while C6 cells possess a normal male karyotype. C6V-1 and C6 cells were employed for characterization of a receptor-adenylate cyclase system of the surface membrane. C6V-1 cells showed lower adenylate cyclase activity than that of C6 cells, though the apparent Km for ATP in both types of cells was the same. The maximal stimulation of adenylate cyclase by isoproterenol was significantly reduced, and Kact for isoproterenol was approximately 18-fold lower in C6V-1 cells. When the concentration of beta-adrenergic receptors was measured by various concentrations of [3H] dihydroalprenolol (DHA), the maximal binding sites of C6 and C6V-1 cells were 760 and 230 fmol/mg protein, respectively, without any changes in the association constant for DHA. The concentration of isoproterenol required for 50% displacement of the [3H] DHA binding (Kd) was the same (around 1.5 X 10(-6)M) in both cells, measured in the presence of GTP. Thus the 19-fold drop in the Kd/Kact ratio in C6V-1 cells suggests an incomplete coupling of beta-receptors to adenylate cyclase. Cyclic AMP phosphodiesterase activity and cAMP content in C6V-1 were lower than in C6 cells. Mitochondrial monoamine oxidase and cytosomal enolase activities, however, were somewhat higher in C6V-1 cells. The results indicate that a set of changes in the receptors and in the cyclic AMP system of C6V-1 is one of the specific alterations by transformation, even though those may not be the cause of cell transformation.