Characteristics of the beta-adrenergic adenylate cyclase system of developing rabbit bone-marrow erythroblasts.

After fractionation of rabbit bone marrow into dividing (early) and non-dividing (late) erythroid cells, the adenylate cyclase activity of membrane ghosts was assayed in the presence of guanine nucleotides ((GTP and its analogue p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate))), the beta-adrenergic agonist L-isoprenaline (L-isoproterenol) and the antagonist L-propranolol. Both GTP and p[NH]ppG increased the adenylate cyclase activity of early and late erythroblasts, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing bone-marrow cells. The effect of L-isoprenaline was completely inhibited by the antagonist L-propranolol, confirming that the response was due to stimulation of beta-adrenergic receptors on the plasma membrane. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind the selective ligand [125I]iodohydroxybenzylpindolol with almost equal affinities, the apparent dissociation constants, Kd, being 0.91 X 10(-8)M and 1.0 X 10(-8) M respectively. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p[NH]ppG during erythroblast development was observed: the dissociation constants of both guanine nucleotides were almost identical with early and late erythroblast membrane preparations [2-3 (X 10(-7) M]. With dividing cells, however, in the presence of L-isoprenaline the dissociation constants of GTP and p[NH]ppG were lower (6 X 10(-8) M). The dose-response curves for isoprenaline competition in binding of [125I]iodohydroxybenzylpindolol by dividing cells showed that the EC50 (effective concentration for half maximum activity) value for isoprenaline was higher in the presence of p[NH]ppG. With non-dividing cells the EC50 value for isoprenaline was equal in the presence and in the absence of p[NH]ppG and similar to that observed with dividing-cell membranes in the presence of the nucleotide. Thus differentiation of rabbit bone-marrow erythroid cells seems to be accompanied by uncoupling of the beta-adrenergic receptors from the adenylate cyclase catalytic protein as well as by a decrease in the number of receptors per cell, but not by changes in the catecholamine and guanine-nucleotide-binding affinities.     

Expression of genes for metabolism of cyclic adenosine 3':5'-monophosphate in somatic cells. beta-Adrenergic and PGE1 receptors in parental and hybrid cells.

Using the ligands [125I]iodohydroxybenzylpindolol and [3H]prostaglandin E1 ([3H]PGE1), we have studied the relationship of receptors for beta-adrenergic agents and for PGE1 to adenylate cyclase in membranes of parental, hybrid, and variant mammalian cell lines. Fusion of parental clones responsive to beta-adrenergic agonists (beta+) with unresponsive clones (beta-) produced hybrid clones with a greatly diminished beta-adrenergic response; beta+ X beta leads to beta-. Binding studies with [125I]iodohydroxybenzylpindolol showed a decreased concentration of beta receptors in six such hybrid clones. Thus, paucity of beta-adrenergic receptors is probably a sufficient, albeit not necessarily complete, explanation for the decreased beta-adrenergic responsiveness of the hybrid clones. When a clone with beta receptor but without apparent adenylate cyclase activity (HC-1) was hybridized with a beta- clone that has adenylate cyclase (B82), a responsive hybrid clone was obtained. In nine cell hybrids produced by the fusion of clones responsive (PGE1+) and unresponsive (PGE1-) to PGE1, high affinity binding sites for [3H]PGE1 were expressed in the same manner as was PGE1-sensitive adenylate cyclase: PGE1+ X PGE1 leads to PGE1+. The chemical specificities and affinities of the parental receptors and responsive adenylate cyclases were faithfully reproduced in the hybrid clones. Activation by PGE1 was proportional to the occupation of the high affinity receptors. In a wild type lymphoma clone (24.3.2), the concentration dependences for binding of [3H]PGE1 and for activation of adenyalte cyclase by PGE1 were identical. In a variant lymphoma clone (94.15.1) lacking adenylate cyclase activity, no high affinity receptors for PGE1 were detected, whereas beta-adrenergic receptors have been demonstrated in this variant clone (Insel, P.A., Maguire, M.E., Gilman, A.G., Coffino, P., Bourne, H., and Melmon, K. (1976) Mol. Pharmacol. 12, 1062-1069). Hybrid cells formed by the fusion of 94.15.1 with cell line RAG (PGE1-) responded to PGE1. Clone 94.15.1 may have receptors for PGE1 of reduced affinity or in low concentration. Alternatively, RAG and 94.15.1 may have complementary genetic defects such that the RAG X 94.15.1 hybrid cells express a hormonally responsive receptor-adenylate cyclase system.     

The effects of ovarian hormones on beta-adrenergic and muscarinic receptors in rat heart

The in vitro and in vivo effects of estrogen and progesterone on muscarinic and beta-adrenergic receptors of cardiac tissue were studied in ovariectomized (OVX) rats. The binding assay for muscarinic receptors was performed under a nonequilibrium condition; whereas the binding assay for beta-adrenergic receptors, under an equilibrium condition. Estrogenic compounds and progesterone were found to have no effect on the binding of the radioligand, [3H]-dihydroalprenolol, to beta-adrenergic receptors in vitro. However, progestins but not estrogenic compounds inhibited the binding of the radioligand, [3H]-quinuclidinyl benzilate, to muscarinic receptors in vitro, with progesterone as the most potent inhibitor (IC50 = 37 microM, apparent Ki = 13 microM). Progesterone was found to decrease the apparent affinity of muscarinic receptors for [3H](-)QNB in vitro. Daily treatment of OVX rats with estradiol benzoate (4 micrograms) or progesterone (2.5 mg) for 4 days had no effect on the muscarinic or beta-adrenergic receptors with respect to the binding affinity and receptor density. However, administrations of these hormones together for 4 days caused an increase in the receptor density of muscarinic receptors without a significant effect on their apparent binding affinity; also these hormones induced a decrease in the binding affinity and an increase in the receptor density of beta-adrenergic receptors. The results of this study demonstrate that progestins are capable of interacting with the cardiac muscarinic receptors in vitro, and indicate that estrogen and progesterone have a synergistic effect to increase the receptor densities of muscarinic and beta-adrenergic receptors as well as to cause a decrease in the binding affinity of beta-adrenergic receptors in vivo.     

Down-regulation of rat liver beta-adrenergic receptors by cysteine

Down-regulation of hepatic beta-adrenergic receptors was indicated by a 56% decrease in the specific activity of 125I-iodocyanopindolol bound to rat liver membrane preparations from rats fed diets containing 15% of casein supplemented with cysteine, instead of methionine or unsupplemented. Down-regulation of hepatic beta-adrenergic receptors by cysteine appears to be mediated through an effect of cysteine on the tissue concentration of S-adenosyl methionine (SAM). The liver tissue concentration of SAM in rats fed cysteine-supplemented diets decreased 53% compared to those fed diets supplemented with methionine. The decrease in liver SAM in rats fed the diet supplemented with cysteine appears to reflect a non-competitive inhibition of methionine adenosyl-transferase by cysteine. Lineweaver-Burk plots demonstrated a dose-related Vmax response to cysteine but did not change the apparent Km at any concentration tested.     

A potential role for catecholamines in the development and progression of carcinogen-induced mammary tumors: hormonal control of beta-adrenergic receptors and correlation with tumor growth.

In order to gain further knowledge on the beta-adrenergic receptor system in DMBA-induced rat mammary tumors, we have studied the correlation between changes in tumoral beta-adrenergic receptor concentration and distribution, progesterone receptor status and tumor growth after ovariectomy and treatment with various ovarian and adrenal steroids, or induction of hyperprolactinemia. Autoradiographic localization of beta-adrenergic receptors in ovariectomized (OVX) animals shows very weak labeling with [125I]cyanopindolol. In these tumors, the connective tissue is predominant, while the epithelial cell content is very low. Similarly, when direct measurements of [125I]cyanopindolol are performed with membrane preparations, beta-adrenergic receptor concentration is sharply reduced 2-3 weeks following ovariectomy or treatment with LHRH against [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide. This effect on the beta-adrenergic receptor population in the tumor is accompanied by the well known effect of castration on tumor growth and progesterone receptor levels, namely a marked regression of tumor growth and a significant decrease in progesterone receptor concentration. Treatment of OVX rats with 17 beta-estradiol (E2) alone or in combination with progesterone (P) caused a highly significant increase in beta-adrenergic and progesterone receptor levels, as well as tumor growth. A similar sharp increase in the value of the three parameters studied was observed following daily treatment of OVX rats with dehydroepiandrosterone (DHEA) or androst-5-ene-3 beta,17 beta-diol (5-ene-diol). The autoradiographic localization of beta-adrenergic receptors in OVX rats treated with 5-ene-diol showed that the epithelial cells were numerous with a high degree of labeling. On the other hand, treatment of OVX animals with the androgen dihydrotestosterone (DHT) did not produce significant changes in beta-adrenergic receptor levels or tumor growth. Finally, endogenously-induced hyperprolactinemia by implanting three anterior pituitary glands under the kidney capsule of OVX animals resulted in a significant increase in beta-adrenergic and progesterone receptor levels as well as tumor growth. The positive correlation observed between changes in beta-adrenergic receptor concentration, progesterone receptor levels and tumor growth indicates a high sensitivity of the beta-adrenergic receptor population of DMBA-induced rat mammary tumors to the hormonal milieu, and suggests that the beta-adrenergic receptor system may represent a valuable parameter of hormone responsiveness.     

Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells.

The beta-adrenergic catecholamine isoproterenol produces a large, rapid, but often a transient, elevation in cellular content of cyclic AMP. We have used the S49 mouse lymphoma cell line, in which genetic variants with specific defects in the pathway of cyclic AMP generation and function have been isolated, to study the increase and subsequent decrease in cyclic AMP levels (termed refractoriness) following incubation of cells with isoproterenol. In wild type S49 cells, isoproterenol produces a peak response in the cellular content of cyclic AMP within 30 min, but the cyclic AMP level falls rapidly thereafter, approaching basal levels by 6 h. Neither inactivation of the drug nor secretion of a nonspecific inhibitor of adenylate cyclase appears to account for the refractoriness. Because isoproterenol refractory cells can still be stimulated by cholera toxin, refractoriness to isoproterenol does not represent a generalized decrease in cellular cyclic AMP response. Particulate preparations from refractory cells have a selective loss of isoproterenol-responsive adenylate cyclase activity, but their activation constants and stereoselectivity for (-)- and (+)-isoproterenol are unaltered. In addition, refractory cells have decreased specific binding of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol. This decrease appears to represent a reduction in the number, but not the affinity, of beta-adrenergic receptor sites. Similar studies in an S49 clone that lacks the enzyme cyclic AMP-dependent protein kinase yield essentially identical findings. Because kinase-deficient cells do not induce the cyclic AMP-degrading enzyme phosphodiesterase after the cellular content of cyclic AMP is increased, induced of phosphodiesterase cannot account for refractoriness to isoproterenol. Cyclic AMP-dependent protein kinase does not appear to be required for either the decrease in beta-adrenergic receptors and isoproterenol-responsive adenylate cyclase, nor does it appear to be required for the development of refractoriness to isoproterenol. In contrast, an S49 clone lacking hormone-responsive adenylate cyclase activity but retaining beta-adrenergic receptors does not appear to lose receptors after being incubated with isoproterenol, either alone or together with dibutyryl cyclic AMP. Therefore, in this clone, receptor occupancy alone or in combination with elevated cyclic AMP levels is insufficient to cause refractoriness. Refractoriness thus appears to require intact adenylate cyclase. This suggests that adenylate cyclase may exert regulatory controls on beta-adrenergic receptors in addition to generation of cyclic AMP.     

Beta-adrenergic agonists and hypertrophy of skeletal muscles.

Chronic administration of some beta-adrenergic agonists markedly stimulates hypertrophy of skeletal muscles. It appears that type II fibers are more responsive to beta-adrenergic agonists than type I fibers. The hypertrophic effect of beta-adrenergic agonists is transient, with the effect diminishing during prolonged treatment. Similarly, some cellular responses including the increase in RNA concentration and the decrease in calpain I activity are also short-lived. Recent evidence suggests that the temporal response is associated with decreased beta-adrenoceptor density. Both increased rate of protein synthesis and/or decreased protein degradation have been suggested as the mechanism of action of these compounds on hypertrophy of skeletal muscles. It is important to consider the temporal nature of cellular responses to chronic treatment of beta-adrenergic agonists as well as the differential effects of these compounds on protein metabolism among skeletal muscle fiber types when investigating the mechanism(s) of action of these compounds.     

Characteristics of flutamide action on prostatic and testicular functions in the rat

The effect of daily treatment with the pure antiandrogen Flutamide has been studied either alone or in combination with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide (LHRH-A), on testicular and prostatic functions in adult male rats. Treatment for 10 days with Flutamide (5 mg/rat, twice daily) caused a marked stimulation of plasma testosterone (T) associated with a significant increase in plasma gonadotropin concentrations and inhibited plasma PRL levels. Testicular weight is not changed following antiandrogen administration but testicular LH/hCG receptor levels are markedly decreased with no change in FSH receptor levels. Moreover, Flutamide treatment alone produces an important inhibition of ventral prostate and seminal vesicle weights associated with a significant decrease in prostatic beta-adrenergic receptor levels but no change is observed in specific ornithine decarboxylase (ODC) activity. Daily LHRH-A treatment at the dose of 1 microgram/day for 10 days decreases plasma T to levels comparable to those found in orchiectomized men (0.30 +/- 0.5 ng/ml). This effect is associated with an almost complete loss of testicular LH/hCG receptors, a decrease in testicular weight, a significant increase in plasma gonadotropins and a marked inhibition of plasma PRL concentration. A relatively smaller inhibition of ventral prostate and seminal vesicle weights follows treatment with the LHRH agonist alone, this effect being accompanied by a significant reduction in beta-adrenergic receptor concentration but no change in prostatic ODC activity. Combination of the two drugs, however, caused a potent inhibitory effect on both ventral prostate and seminal vesicle weight to values similar to those found in castrated rats. The prostatic weight loss is accompanied by a marked fall in ODC activity and in the concentration of beta-adrenergic receptors. The present data clearly show that combined treatment with an LHRH agonist and a pure antiandrogen is highly effective in inhibiting, not only prostatic growth, but also two androgen-sensitive parameters of prostatic activity.     

Effect of the Tricyclic Antidepressant Desipramine on β-Adrenergic Receptors in Cultured Rat Glioma C6 Cells

Rat glioma C6 cells, cultured in the presence of the tricyclic antidepressant desipramine, lost a significant number of beta-adrenergic receptors in a time- and dose-dependent manner. A similar loss was observed whether binding was determined on intact cells with the hydrophilic beta-adrenergic antagonist (+/-)-[3H]4-(3-tert-butylamino-2-hydroxypropoxyl)benzimidazole-2-o n HCl ([3H]CGP-12177) or on cell lysates with the more hydrophobic antagonists [125I]iodocyanopindolol or [3H]dihydroalprenolol. When stimulated with the agonist isoproterenol, desipramine-treated cells accumulated less cyclic AMP than control cells. The affinity of the beta-adrenergic receptors for either antagonist or agonist was unchanged after desipramine treatment. Desipramine interacted only weakly with the receptors and competed for [125I]iodocyanopindolol binding with a Ki of 30 microM. The presence in the culture medium of alprenolol or propranolol, potent beta-adrenergic antagonists, however, did not prevent the reduction in receptors by desipramine. Desipramine also caused a loss of beta-adrenergic receptors from cells maintained in serum-free medium and the cells themselves did not contain or secrete endogenous catecholamines. Although desipramine is a potent inhibitor of catecholamine uptake, it appears unlikely that the observed loss of beta-adrenergic receptors in rat glioma C6 cells exposed to the drug is due to an increase in extracellular catecholamine levels or to a direct interaction with the receptors.     

Identification of beta-adrenergic receptors in pulmonary alveolar type II cells

Specific beta-adrenergic receptors have been identified in dissociated preparations of rabbit lung cells greatly enriched for alveolar type II cells and compared with receptors in preparations of mixed lung cells and erythrocytes. Freshly isolated type II cells as well as mixed dissociated lung cells and erythrocytes from fetal (28 days gestation) and adult rabbits contained high-affinity, low-capacity binding sites for [3H]dihydroalprenolol (DHA). Binding to all preparations was stereospecific and characteristic of the beta 1-subtype of beta-adrenergic receptors. The concentrations of the receptors were similar in mixed lung cells and alveolar type II cells, indicating that beta-adrenergic receptors are present not only in type II cells but also in other lung cell types. When the contribution of erythrocytes to receptor concentration observed in type II cells was determined, it was found to be insignificant. In mixed lung cells, both the affinity and concentration of the receptors were higher in adult than fetal preparations. The affinity of the receptors was also higher in adult than fetal type II cells, although we did not find a significant age-related difference in receptor concentrations in this cell type. These results suggest that stimulation of surfactant secretion observed after exposure of lung tissue to beta-adrenergic agonists is mediated by specific beta-adrenergic receptors on alveolar type II cells.     

Guanine nucleotides modulate the affinity of antagonists at beta-adrenergic receptors

Investigation of the properties of the binding of the radiolabelled antagonists (125I)-iodohydroxybenzylpindolol, (125I)-iodopindolol, and (125I)-iodocyanopindolol to beta-adrenergic receptors of L6 myoblast membranes revealed that guanine nucleotides caused a 2 to 4.5 fold increase in the apparent affinity of these antagonists. No significant effects of GTP were observed on the density of binding sites determined with each radioligand. GTP, GDP, and GMPPNP were of similar high affinity in producing this effect, while GMP was much less potent, and ATP was without effect. Under similar assay conditions GTP reduced the apparent binding affinity of the agonist isoproterenol for the beta-adrenergic receptors of L6 cells. The results indicate that, contrary to previous observations, guanine nucleotides affect not only the interactions of agonists with beta-adrenergic receptors, but also the interaction of antagonists with these adenylate cyclase-linked receptors.     

Determination of the desensitization of beta-adrenergic receptors by [3H]CGP-12177.

Isoprenaline treatment of C6-glioma cells induced a fast decrease in the number of beta-adrenergic receptors as determined by binding of [3H]CGP-12177, which paralleled the decrease in the hormonally stimulated adenylate cyclase activity. The total number of receptors, as determined by binding of (-)-[3H]dihydroalprenolol, did not decrease. Separation of the beta-adrenergic receptors on a sucrose density gradient showed that the decrease in the number of receptors detectable with CGP-12177 was due to a movement of the receptors from the plasma membrane to a vesicular cell compartment. By using both (-)-[3H]dihydroalprenolol and [3H]CGP-12177 it is thus possible to differentiate between the total number of receptors and those present at the plasma membrane in an unfractionated cell lysate.     

Beta-adrenergic receptor concentration and subtype in the corpus luteum of the adult pseudopregnant rat

Luteal beta-adrenergic receptor concentration and subtype were determined in adult pseudopregnant rats during and after the period of the functional luteal phase. The specific beta-adrenergic receptor ligand (-)-3-[125I]iodocyanopindolol ([125I]ICYP) was used to determine the receptor concentration in corpora lutea of adult pseudopregnant rats. A 3-fold increase in beta-adrenergic receptor concentration was seen during the first 2-3 days of pseudopregnancy, whereafter the receptor concentration declined. During the functional luteal regression period (Day 12-15) the receptor levels were still low. In regressed (Day 16-22) corpora lutea a temporary increase in beta-receptor concentration was seen which may represent some role for beta-adrenergic mechanisms in the regulation of morphological regression in the corpus luteum. To determine the beta-adrenergic subtype, competition of [125I]ICYP-binding with selective beta 1- and beta 2-adrenergic antagonists was assessed in corpora lutea of different ages and in rat heart and uterus. The beta-adrenergic receptors in corpora lutea of adult pseudopregnant rats were shown to be solely of the subtype beta 2, regardless of the luteal age.     

Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian beta-adrenergic receptor.

A series of mutant avian beta-adrenergic receptors with progressively truncated carboxyl termini have been expressed in insect and mammalian cells. Removal of 18-124 amino acid residues caused multiple phenotypic changes in the receptor. Membranes from cells that expressed the truncated receptors displayed elevated basal (2- to 3-fold) and agonist-stimulated adenylylcyclase activities. Adenylylcyclase activity in these membranes also displayed greater stimulation in response to partial agonists. Activity was also markedly stimulated by beta-adrenergic ligands that are usually considered to be antagonists (alprenolol, greater than 4-fold; propranolol, approximately 2-fold). Wild type receptor did not mediate a response to these classical antagonists. After purification and reconstitution with Gs, the truncated receptors did not appear to be more active than the wild type. Guanine nucleotides modulated the affinity of agonist for the truncated receptors, whereas the affinity of agonist for the wild type receptor was not altered by guanine nucleotides. The truncated receptors were solubilized from the membrane more efficiently and were more susceptible to amino-terminal proteolysis than was the wild type protein. These results suggest interaction of the carboxyl terminus of the avian beta-adrenergic receptor with cellular regulatory or structural elements.     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities of S49 lymphoma cells by agents increasing cyclic AMP

We have examined the regulation of two key enzymes that control polyamine biosynthesis-L-ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) - by agents increasing cAMP in S49 lymphoma cells. Incubation of wild type S49 cells with beta-adrenergic agonists (terbutaline or isoproterenol) inhibited ODC and SAMDC activities rapidly (less than 2 hr). more quickly than these agents arrested the cells in the G1 phase of the cell cycle. The beta-adrenergic antagonist propranolol blocked inhibition of ODC activity produced by isoproterenol, but only if added simultaneously or less than 4 hr after the agonist. Incubation of wild type S49 cells with cholera toxin or PGE1 also inhibited ODC activity. Decreases in ODC activity produced by beta-adrenergic agonists, cholera toxin, PGE1 or dibutyryl cAMP were all enhanced by the phosphodiesterase inhibitor Ro 20-1724. Results of studies of ODC and SAMDC activity in S49 variants having lesions in the pathway of cAMP generation and action were as follows: kin- cells (which lack cAMP-dependent protein kinase activity) showed no inhibition of ODC by any agent; AC- cells (which have absent nucleotide coupling units in their adenylate cyclase system) only demonstrated inhibition in response to dibutyryl cAMP; UNC cells (which have deficient coupling of hormone receptors and adenylate cyclase) only demonstrated inhibition in response to dibutyryl cAMP and cholera toxin, and beta-depleted cells (which have a decreased number of beta-adrenergic receptors) responded as did wild type cells except for absent response to isoproterenol. We conclude that inhibition of ODC and SAMDC activity in S49 cells is an early response to agents that increase cAMP and that this action occurs via the "classical" pathways of activation of adenylate cyclase and protein kinase. These results in S49 cells contrast with evidence in other systems in which cAMP has been suggested to enhance polyamine biosynthesis, perhaps through alternative mechanisms.     

Agonist Interactions with Beta-Adrenergic Receptors Following Chronic Administration of Desipramine or the Atypical Antidepressants,Iprindole and Mianserin

Abstract

Desipramine (DMI), decreased the maximum number of beta-adrenergic receptors by approximately 10, 20, 30, and 20% in groups of rats treated i.p. with 5 mg/kg for 14 days or 10 mg/kg for 7, 14, or 21 days, respectively. In studies of agonist competition for beta-adrenergic receptors labelled with [¹²⁵I]-CYP, chronic DMI administration caused a selective decrease in those receptors normally found in the high affinity conformation in proportion to the dose of DMI administered. No change was observed in either the number of receptors in the agonist low affinity conformation or in the affinity of any drug for the high or low affinity conformations of the receptors. Therefore, chronic DMI caused a selective decrease in the beta-adrenergic receptors linked to adenylate cyclase but did not appear to change other properties of the receptors that would be manifested as a change in their ability to interact with adrenergic agonists. Neither iprindole (15 mg/kg i.p., 14 days) nor mianserin (10 mg/kg i.p., 14 days) decreased the number of receptors, the proportions of agonist high or low affinity receptors, or the affinity of competitor drugs for these receptors, suggesting a different mechanism for the reported loss of adenylate cyclase activity following these drugs than the down-regulation of receptors observed with chronic DMI treatment.     

Reciprocal expressions of alpha 1- and beta-adrenergic receptors, but constant expression of glucagon receptor by rat hepatocytes during development and primary culture

The stimulations of cyclic AMP formation and adenylate cyclase activity by glucagon and isoproterenol were both found to be highest in neonatal rat hepatocytes and to decrease during development. Adult hepatocytes still showed a considerable response to glucagon, but a negligible response to isoproterenol. The decrease in cyclic AMP formation during development can be explained in the case of the response to beta-adrenergic agonist as due to decrease of its receptor number, judging from binding of [125I]iodocyanopindolol to purified plasma membranes. But in the case of the glucagon response, the decrease in the response may be due to change of post-receptor components of the adenylate cyclase system, because the receptor number tended to increase during development, as shown by binding of [125I]iodoglucagon. Similarly, alpha 1-adrenergic receptors increased in number during development, but their IC50 value did not change, as measured by binding of [3H]prazosin to plasma membranes. Previous studies on primary cultures of adult rat hepatocytes showed that the beta-adrenergic response and its receptor number increased markedly during short-term culture (Nakamura, T., Tomomura, A., Noda, C., Shimoji, M., & Ichihara, A. (1983) J. Biol. Chem. 258, 9283-9289). However, in this work the amount of alpha 1-adrenergic receptor of adult rat hepatocytes was found to decrease by one third during 1-2 days culture. Therefore, changes of alpha 1- and beta-adrenergic receptors during development of rat liver and during primary culture of adult rat hepatocytes were reciprocal, although the directions of change in the two conditions were opposite. The additions of various hormones to primary cultures of adult rat hepatocytes did not affect the reciprocal changes of adrenergic receptors, suggesting that these hormones did not regulate the changes of the receptors.     

Adrenergic receptors and adenylate cyclase activity in hepatocytes of the streptozotocin-diabetic rat.

Effects of short and long exposure to the diabetic state induced by an injection of streptozotocin to young female rats on glucagon- and catecholamine-sensitive adenylate cyclase activity and adrenergic receptors of hepatic membranes have been studied. The short period of exposure to the diabetic state exhibited an increase in the sensitivity of the enzyme to isoproterenol without changes in the affinity and the number of beta-adrenergic receptors. The increased response of adenylate cyclase activity to isoproterenol was accompanied with a greater GTP-induced lowering of the affinity to the beta-adrenergic agonist in diabetic membranes than in the controls. The chronic diabetic state produced a decrease in the adenylate cyclase activity to hormonal or non-hormonal stimuli with a fall in the number of alpha- and beta-adrenergic receptors. These results suggest that the observed effects of the diabetic state on hormonally sensitive adenylate cyclase activities and their receptor binding sites of the hepatic membranes would vary depending on the duration and/or severity of the diabetic state experimentally induced.     

Adrenergic reactions of sheep rumen in vivo

Pharmacodynamical analysis of alpha-adrenergic and beta-adrenergic reactions of sheep rumen was performed in vivo after administration of agonists and antagonists of alpha-adrenergic and beta-adrenergic receptors. It was found that phenylephrine, and in a lower degree propranolol, stimulated the motor activity of sheep rumen, while adrenaline, noradrenaline, isoprenaline, phenoxybenzamine and regitine depressed this activity. Propranolol abolished atonia produced by catecholamines in sheep rumen, and previous blockade of beta-adrenergic receptors with propranolol reversed the relaxing action of catecholamines on the muscular elements in the rumen. The experiments in vivo confirmed the adrenergic effects on the motor activity of the rumen of sheep obtained in earlier investigations on isolated muscles of the rumen. It is suggested that noradrenaline exerts an ambireceptro effect (similar to that of adrenaline) on the motor activity in sheep rumen.