Catecholamine-stimulated Adenylate Cyclase Activity Research Articles

Functional desensitisation of beta-adrenergic receptors of avian erythrocytes by catecholamines and adenosine 3',5'-phosphate.

Prolonged exposure to beta-adrenergic agonists of pigeon erythrocytes causes a reversible loss (70%) of catecholamine-stimulated adenylate cyclase activity without reduction in the number of beta-adrenergic receptors. In addition a less pronounced decrease in non-stimulated and NaF-stimulated adenylate cyclase activity (15-22%) is observed, appearing at different agonist concentrations and at a different rate. Dibutyryladenosine 3',5'-phosphate and the phosphodiesterase inhibitor methylisobutylxanthine partially mimick the action of the beta-adrenergic agonist, thus pointing to a possible role of adenosine 3',5'-phosphate in establishing desensitization. When adenylate cyclase from desensitized cells is stimulated with 5'-guanylyl-imidodiphosphate in the presence or absence of catecholamines the lag period preceding the attainment of maximal activity is extended. Likewise the rate of reversal by GTP or GTP of persistent activation of adenylate cyclase is slowed down. This is therefore interpreted to mean that the loss in hormonal stimulation on treatment of pigeon red blood cells with beta-adrenergic agonists is due to a delayed exchange of GDP against GTP on the regulatory GTP-binding protein. Furthermore, we conclude that events causing the refractory state in avian erythrocytes should occur at a site distal to the beta-adrenergic receptor.

Leptin affects adenylate cyclase activity in H9c2 cardiac cell line: effects of short- and long-term exposure

Leptin has been hypothesized to be a pathophysiologic link between obesity and cardiovascular diseases. Because the adenylate cyclase (AC) system is a main effector of β-adrenergic receptors and leptin has been shown to modulate AC activity in other cell lines, a leptin impact on cardiac AC activity was hypothesized. Therefore, acute and chronic effects of leptin on a rat cardiac cell line (H9c2) were investigated. Leptin affected both basal (+13% at 30 min and −16.4% after 18 h v untreated cells) and catecholamine-stimulated AC activity (isoproterenol + leptin at 30 min or 18 h was +21% v untreated cells; norepinephrine + leptin at 30 min was +38.8% v untreated cells; and norepinephrine + leptin at 18 h was +6% v untreated cells). Thus, long-term leptin treatment was associated with a reduced AC activity and a different responsiveness to catecholamines. The AC activity on leptin treatment was accompanied by changes in levels of proteins structurally or functionally related to AC complex (AC, Gαs, Gαi, p21-ras). These data indicate that the AC complex is profoundly affected at more than one level by leptin treatment in the H9c2 cardiac cell line. Differences in AC activity after short- and long-term exposure to leptin and the interaction between leptin and catecholamine might provide further insight to the understanding of the development of hypertension and congestive heart failure in obese patients.

Interactions of Esmolol and Adenosine in Atrioventricular Nodal-Dependent Supraventricular Tachycardia: Implication for the Cellular Mechanisms of Adenosine

Introduction: Cellular mechanisms of adenosine include a direct effect on the activation of the adenosine-sensitive potassium current (I_K,Ado) and an indirect effect on antagonism of catecholamine-stimulated adenylate cyclase activity. However, previous studies evaluating the influence of catecholamine activity on the electrophysiologic effects of adenosine have yielded conflicting results. We tested the hypotheses that if adenosine exerts its atrioventricular (AV) nodal blocking effects directly by activating the I_K,Ado potassium current, rather than indirectly by reversing the catecholamine effects, then pretreatment with β-adrenergic blockade would not potentiate the effects of adenosine in terminating AV nodal-dependent supraventricular tachycardia (SVT). Methods and Results: During sustained AV nodal reentrant tachycardia (AVNRT) or AV reentrant tachycardia (AVRT) in 28 patients, adenosine was rapidly injected in incremental doses of 1.5, 3, 6, 9, 12 and 18 mg to determine the lowest effective dose required for tachycardia termination before and immediately after the end of esmolol infusion. Esmolol infusion was started with loading doses of 500 µg/kg/min for 1 min and 150 µg/kg/min for 4 min, followed by a maintenance infusion of 50–100 µg/kg/min. Esmolol infusion was continued until the tachycardia was terminated or the maximal dose of 100 mg was reached. Adenosine was effective in terminating SVT in all 28 patients with a mean lowest effective dose of 96 ± 54 µg/kg before esmolol. During esmolol infusion, tachycardia was reproducibly terminated in 8 patients (6 with AVNRT, 2 with AVRT) with a mean dose of 67 ± 23 mg. In the other 20 patients with persistent tachycardia after 100 mg of esmolol infusion, the lowest effective dose of adenosine could be determined in 19 patients. In the remaining patient with AVRT, the maximal dose of adenosine (18 mg) was unable to terminate the tachycardia immediately after the end of esmolol infusion. In these 19 patients, esmolol infusion caused significant lengthening of the tachycardia cycle length from 338 ± 36 to 372 ± 51 ms (p < 0.0001) and reduction of the mean arterial blood pressure from 96 ± 15 to 88 ± 18 mm Hg (p = 0.034). Compared to the dosage that was determined before esmolol infusion, the lowest effective dose of adenosine remained the same in 13 patients after the end of esmolol infusion, whereas the dose increased in 5 and decreased in 1 patient. The mean lowest effective dose of adenosine was not significantly different before (98 ± 54 µg/kg) and immediately after (115 ± 56 µg/kg) the end of esmolol infusion (p = 0.054). Conclusions: Intravenous esmolol infusion (up to 100 mg total dose) usually fails to terminate AV nodal-dependent SVT. In the esmolol-resistant tachycardia, esmolol pretreatment does not produce a positive synergistic effect on the efficacy of adenosine-induced termination of SVT. Therefore, in this tachycardia adenosine may exert its effects on AV nodal conduction directly by activation of the I_K,Ado potassium current, rather than by antagonizing the β-adrenergic system.

Cardiac membrane lipid composition and adenylate cyclase activity following dietary eicosapentaenoic acid supplementation in the marmoset monkey

The effects of dietary eicosapentaenoic acid (20:5(n-3)) on cardiac membrane phospholipid fatty acid composition and membrane-associated adenylate cyclase activity were investigated in the marmoset monkey. A 20:5(n-3) concentrate (as the ethyl ester) was tested with high fat diets formulated to give a polyunsaturated:monounsaturated:saturated (P:M:S) fatty acid ratio of either 1:1:1 (control diet, no added cholesterol) or 0.1:0.6:1.0 (atherogenic-type diet, 0.2% cholesterol) that were fed for 30 weeks. The various dietary lipid treatments did not alter the proportions of the major phospholipids present in cardiac membranes. Both 20:5(n-3)-supplemented diets resulted in an increase in the proportions of 20:5(n-3) and 22:5(n-3), but not 22:6(n-3) in membrane phospholipids and a decrease in the proportion of 18:2(n-6) and 20:4(n-6). Greater incorporation of the n-3 polyunsaturated fatty acids occurred when the level of dietary linoleic acid was reduced. These changes in fatty acid profiles were evident to a greater extent in phosphatidylcholine and phosphatidylethanolamine in comparison with cardiolipin and sphingomyelin. These diets did not influence catecholamine-stimulated adenylate cyclase activity in terms of sensitivity or responsiveness to isoproterenol. The response of adenylate cyclase to other agonists such as epinephrine, norepinephrine, sodium fluoride, and forskolin was also unaffected by the dietary lipid treatments. The results clearly show that there is extensive incorporation of dietary 20:5(n-3) and its elongation product 22:5(n-3) into cardiac membrane phosphatidylcholine and phosphatidylethanolamine. However, unlike the reported effect of altered cardiac membrane cholesterol status, alterations in cardiac membrane phospholipid fatty acid composition alone appear not to affect membrane-associated adenylate cyclase activity in the marmoset monkey.

Adenosine receptor coupling to adenylate cyclase of rat ventricular myocyte membranes

The effects of adenosine analogues on beta-adrenergic receptor and receptor-independent elicited increases in adenylate cyclase activity were investigated using membranes obtained from primary cultures of adult rat ventricular myocytes. Phenylisopropyladenosine, an A1-receptor agonist, at concentrations of 0.1, 1.0, and 10 microM, maximally inhibited isoproterenol-stimulated adenylate cyclase activity by 35, 55, and 41%, respectively. The inhibition by phenylisopropyladenosine was antagonized by 10 microM theophylline. One micromolar phenylisopropyladenosine was much less effective at attenuating forskolin-stimulated activity, such that the maximum inhibition was 26%. Phenylisopropyladenosine had no effect on adenylate cyclase stimulation by 5'-guanylylimidodiphosphate. Phenylaminoadenosine, an A2 agonist, at 10 microM or greater stimulated adenylate cyclase activity. This effect was not significantly inhibited by theophylline or 0.1 microM 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), which antagonized phenylisopropyladenosine inhibition of isoproterenol-stimulated adenylate cyclase activity. Additionally, N-ethylcarboxamidoadenosine, a nonselective adenosine-receptor agonist, had no effect on adenylate cyclase activity in the absence of DPCPX but stimulated adenylate cyclase activity in the presence of DPCPX. These results indicate that both A1 and A2 receptors exist on the ventricular myocyte sarcolemma. More importantly, the findings suggest that adenosine inhibition of catecholamine-stimulated adenylate cyclase activity is primarily due to an alteration in beta-adrenergic receptor-mediated transduction and perhaps in part by a direct inhibition of the catalytic component.

A Different Desensitization Pattern of Cardiac β-Adrenoceptor Subtypes by Prolonged In Vivo Infusion of Isoprenaline

(-)Isoprenaline was continuously administered to rats at a rate of 0.4 mg/kg/h for 7 days via subcutaneously (s.c.) implanted osmotic minipumps. This treatment induced cardiac hypertrophy and a marked decrease in basal as well as catecholamine-stimulated adenylate cyclase activity in a ventricular plasma membrane fraction. The total number of beta-adrenoceptors was downregulated by one-half the amount of the receptor sites obtained in a control group. However, in the isoprenaline-treated group, the beta 2-adrenoceptors constituted a significantly smaller proportion of the total beta-adrenoceptor population (28%) than in the control group (50%). Transformation of these relative into absolute values indicates that prolonged isoprenaline treatment induced a significantly higher downregulation of beta 2- than of beta 1-adrenoceptors. The fact of a different beta-adrenoceptor desensitization pattern in response to in vivo administration of nonselective beta-adrenergic agonists therefore must be taken into consideration when desensitization is used as a method for determination of subtype selectivity of an agonist per se. However, we were unable to detect the "lost" beta-adrenoceptors in a light vesicular fraction. In our study, this fraction was not separable from plasma membranes, as substantiated by levels of plasma membrane markers as high as in the plasma membrane fraction and by a guanine nucleotide-dependent adenylate cyclase activity.

Beta-adrenergic receptors in hamster smooth muscle cells are transcriptionally regulated by glucocorticoids.

Steroid hormones modulate adrenergic receptor responsiveness and receptor number. To investigate the regulation of the beta 2-adrenergic receptor gene by glucocorticoids we examined the effects of the synthetic glucocorticoid agonist triamcinolone acetonide on the expression of beta 2-adrenergic receptors in DDT1MF-2 hamster smooth muscle cells. Glucocorticoid treatment (1 X 10(-7) M) produced a 2.2 +/- 0.4-fold (n = 8) increase in beta 2-adrenergic receptor number (maximum) between 6 and 12 h) as determined by radioligand binding and a similar increase in catecholamine-stimulated adenylate cyclase activity. Steady-state levels of beta 2-adrenergic receptor mRNA, analyzed by Northern blot hybridization, were increased 2.4 +/- 0.4-fold (n = 6) within 1 h, while actin mRNA levels were unchanged throughout the experiment. These steroid-induced increases in beta 2-adrenergic receptor mRNA returned to control levels by 24 h and were followed by a much slower decline in beta 2-adrenergic receptor in plasma membranes. The rate of beta 2-adrenergic receptor gene transcription, assessed by nuclear run-off transcription assays, increased 3.1 +/- 0.1-fold (n = 2) in cells treated for 30 min with 1 X 10(-7) M triamcinolone acetonide. These studies indicate that glucocorticoids regulate the beta 2-adrenergic receptor-adenylate cyclase system by controlling the rate of transcription of the beta 2-adrenergic receptor gene and hence the responsiveness of the enzyme to catecholamine stimulation.

The influence of dietary lipid supplementation on cardiac β-adrenergic receptor adenylate cyclase activity in the marmoset monkey

Dietary lipid supplements high in either saturated fat derived from sheep kidney fat or unsaturated fat derived from sunflower seed oil, and a low mixed fat reference diet were fed to marmoset monkeys for 20 months and the effects on cardiac membrane lipid composition, and myocardial catecholamine-stimulated adenylate cyclase and β-adrenergic receptor binding activity were investigated. For cardiac membranes enriched for β-adrenergic binding activity, the dietary lipid treatment resulted in small changes in the proportion of saturated to unsaturated fatty acids and substantial changes in the ( n - 6) to ( n - 3) series of unsaturated fatty acids in the membrane phospholipids. The sheep kidney fat diet increased the cholesterol-to-phospholipid ratio in cardiac membranes in comparison to the other diets. This diet also significantly elevated basal and isoproterenol-, epinephrine- and norepinephrine-stimulated adenylate cyclase activity. The value of the dissociation constant ( K d ) and the receptor number ( B max ) for the binding of [ 125I]ICYP to the β-adrenergic receptor was significantly reduced in marmosets fed the sheep kidney fat diet. These results suggest that dietary lipids can influence the activity of the β-adrenergic/adenylate cyclase system of the heart. Modulation of this transmembrane signalling system may be induced by changes in the properties of the associated membrane lipids, particularly by alteration in the membrane cholesterol-to-phospholipid ratio. This effect may be limited to those animal species in which the nature of the dietary fatty acid intake may be influencing cardiac membrane cholesterol homeostasis, which is in agreement with previous results in rats following dietary cholesterol supplementation (McMurchie et al. (1987) Biochim. Biophys. Acta 898, 137–153). ICYP, (−)-iodocyanopindolol.

Myometrial desensitization after ritodrine infusion.

We have developed a model in pregnant sheep to investigate pharmacological agents used for suppression of preterm labor. This model allows repetitive determinations of uterine contractility and simultaneous measurements of myometrial receptor and postreceptor events. We used the model to study the beta-adrenergic agent ritodrine. We infused 11 pregnant sheep with ritodrine and 3 with physiological saline for 24 h. Oxytocin boluses were given before and at 5 and 22 h after onset of the infusion. Myometrial biopsies were obtained before and immediately after the infusion. After 22 h of ritodrine infusion, uterine contractility in response to the same oxytocin bolus was 50% greater than at 5 h (P less than 0.02). Myometrial membrane beta-adrenergic receptor density decreased 49% (P less than 0.005), and catecholamine-stimulated adenylate cyclase activity was reduced 70% (P less than 0.005). The model thus demonstrates that use of the beta-adrenergic agonist ritodrine in a clinically relevant manner results in tachyphylaxis of its effects on both physiological parameters and the receptor cascade system.

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and β-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the ( n − 6) to ( n − 3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the β-adrenergic receptor, determined by [ 125I](−)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of β-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated β-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the β-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.

Phenylisopropyladenosine (PIA) inhibits catecholamine-stimulated adenylate cyclase activity in adult rat ventricular myocyte membranes

Phenylisopropyladenosine (PIA) inhibits catecholamine-stimulated adenylate cyclase activity in adult rat ventricular myocyte membranes

Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins.

beta-Adrenergic receptors, the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs), and adenylate cyclase were each purified and reconstituted into unilamellar vesicles composed of phosphatidylethanolamine and phosphatidylserine (3:2, w/w). The molar ratio of receptor:Gs:adenylate cyclase was estimated to be about 1:10:1. Adenylate cyclase activity in the vesicles was stimulated up to 2.6-fold by beta-adrenergic agonists. Stimulation was dependent on the presence of guanine nucleotide, displayed appropriate beta-adrenergic selectivity and stereoselectivity for agonists, and was blocked appropriately by beta-adrenergic antagonists. Therefore, while additional proteins may modulate adenylate cyclase activity in native membranes, these results show that these three proteins are sufficient for the expression of hormone-stimulated adenylate cyclase.

Beta-Adrenergic receptors, adenylate cyclase activity, and cardiac dysfunction in the diabetic rat.

Cardiomyopathy is a complication of human diabetes mellitus. The relationship of cardiac function to the beta-adrenergic receptor and catecholamine-stimulated adenylate cyclase activity was investigated in the streptozotocin-diabetic rat. beta-Adrenergic receptor number in cardiac membranes from diabetic rats was reduced. After 2 weeks of diabetes, the response of adenylate cyclase to isoproterenol stimulation was not altered. Cardiac contractile function assessed by the maximum rate of rise of left ventricular pressure (LV dP/dtmax) in an open-chest anesthetized rat was also unchanged from control at 2 weeks. However, after 4 weeks of diabetes, the sensitivity of adenylate cyclase to isoproterenol stimulation was depressed and abnormalities in cardiac contractility were noted, including a depressed response of LV dP/dtmax to graded isoproterenol infusion. These studies suggest that alterations in beta-adrenergic receptors and their coupling to adenylate cyclase may be important in the development of diabetic cardiomyopathy.

Halothane Inhibition of Canine Myocardial Adenylate Cyclase-Modulation by Endogenous Factors

We have hypothesized that the halothane-induced depression of myocardial contractility can be explained, at least in part, by halothane's depression of adenylate cyclase, previously demonstrated in whole homogenates of myocardial tissue. Canine myocardial sarcolemmal membranes, which contain the adenylate cyclase of myocardial cells, were separated from other cellular constituents. Halothane did not depress catecholamine-stimulated adenylate cyclase activity in this preparation. Reconstitution of the sarcolemmal membrane preparation with a 100,000 X g adenylate cyclase-free supernatant restored the depressant effect of halothane on adenylate cyclase stimulated by guanosine triphosphate (GTP) 100 microM alone (-55%, P less than 0.01) or in combination with l-isoproterenol 1 microM (-38%, P less than 0.05) or 2.5 microM (-40%, P less than 0.01). Dilution of the supernatant to half-strength decreased the magnitude of the halothane-induced depression of adenylate cyclase activity to 19% (P less than 0.01); at one-quarter dilution, the effect was no longer significant. This study demonstrates the presence of endogenous modulators of the action of halothane on canine myocardial adenylate cyclase that can be reversibly separated from the adenylate cyclase complex.

Characterization of an altered membrane form of the beta-adrenergic receptor produced during agonist-induced desensitization.

Incubation of 1321N1 human astrocytoma cells with 1 microM isoproterenol rapidly results in the conversion of a portion of the beta-adrenergic receptors to a membrane form that can be separated from markers for the plasma membrane by sucrose density gradient or differential centrifugation. This "light peak" form of the receptor reaches a maximal level within 10 min of incubation of cells with catecholamine. Two types of experiments suggest that the early phase of catecholamine-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase can be separated into at least two reactions. First, the agonist-induced loss of catecholamine-stimulated adenylate cyclase activity precedes the appearance of beta-adrenergic receptors in the light peak fraction by 1-2 min. Second, pretreatment of cells with concanavalin A prior to induction of desensitization blocks the formation of the light peak form of beta-adrenergic receptors without blocking the "uncoupling" reaction as measured by catecholamine-stimulated adenylate cyclase activity. Specificity for the reaction that converts beta-adrenergic receptors to the light peak form is indicated by the lack of a catecholamine-induced alteration in the sucrose density gradient distribution of muscarinic cholinergic receptors, adenylate cyclase or the guanine nucleotide-binding proteins, Ns and Ni. The light peak of beta-adrenergic receptors migrates at a density similar to that of at least a portion of the activity of galactosyltransferase, a marker for Golgi. Enzyme marker activities for lysosomes and endoplasmic reticulum are not associated with this population of beta-adrenergic receptors. Taken together, these and other data suggest that incubation of 1321N1 cells with isoproterenol results in a rapid uncoupling of beta-adrenergic receptors from adenylate cyclase which is followed by a change in the membrane form of the receptor. This latter step most likely represents internalization of receptors into a vesicular form which may then serve as the precursor state from which receptors are eventually lost from the cell.

Evidence for catecholamine-stimulated adenylate cyclase activity in frog and rabbit corneal epithelium and cyclic AMP-dependent protein kinase and its protein substrates in frog corneal epithelium

Evidence was obtained for catecholamine-stimulated adenylate cyclase activity in particulate fractions of frog and rabbit corneal epithelium. Epinephrine (10 −5 m) stimulated adenylate cyclase by 22 and 53% in the frog and rabbit, respectively. The corresponding changes were statistically significant ( P < 0·01) when the data was analyzed using paired variates. Preincubation with 10 −4 m propranolol eliminated any stimulatory effect by 10 −5 m isoproterenol. Adenylate cyclase activity derived from either source was activated several fold by either 10 m m NaF or 10 −5 mGpp (NH)p. Soluble fractions of homogenized frog corneal epithelium contained cyclic AMP-dependent protein kinase activity which was half-maximally stimulated by about 6 n m cyclic AMP. Evidence was also obtained for the presence of protein substrates of cyclic AMP dependent protein kinase in frog corneal epithelium. With exogenous cyclic AMP and protein kinase, a rapid 32P labelling of proteins having approximate molecular weights of 56, 46, 23 and 21 K was obtained with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. A less marked and slower increase in phosphoprotein formation was observed when corneal membranes were incubated with cyclic AMP in the absence of added protein kinase.

Trophoblastic cells of the hydatidiform mole contain a beta 1-subtype adrenergic receptor.

Both beta 1- and beta 2-adrenergic receptors have been previously described in normal human placental homogenates; the cells upon whose surface membranes these receptors reside have not been identified. In order to show that a beta 1-adrenergic receptor is present on trophoblastic cells, the cells which mediate maternal-fetal transport and produce placental hormones, beta-adrenergic receptors were demonstrated in membrane fractions of human hydatidiform mole. Microscopic sections of the mole samples used demonstrated edematous villi lined by trophoblastic cells with minimal nontrophoblastic (stromal or vascular) contamination compared with placenta. (--)-[3H]Dihydroalprenolol [(--)-[3H]DHA] binding to molar membranes was reversible and saturable to a single class of sites (Kd = 0.97 +/- 0.12 nM; n = 7; maximum binding capacity, 72.9 +/- 6.4 fmol/mg protein). (--)-[3H]DHA binding was associated with catecholamine-stimulated adenylate cyclase activity. Agonist competition for the molar beta-adrenergic receptor showed the order of potency to be (--)isoproterenol much greater than norepinephrine = epinephrine, characteristic of a beta 1-adrenergic receptor subtype. Competition for (--)-[3H]DHA binding to trophoblastic membranes by the beta-adrenergic receptor subtype-specific agents metoprolol (beta 1 selective) and zinterol (beta 2 selective) was also characteristic of a homogeneous subtype of beta 1-adrenergic receptors. Because beta 1-adrenergic receptors alone were seen on trophoblast cells, the beta 2-adrenergic receptor in placenta must reside on nontrophoblastic elements (stromal or vascular endothelium). No differences in beta-adrenergic receptor binding were seen related with ploidy (2 or 3 N), the presence or absence of a fetus, or the progression of the mole to choriocarcinoma. Two choriocarcinoma cell lines, BeWo and JEG-3, however, showed no specific (--)-[3H]DHA binding. Human trophoblast contains beta 1-adrenergic receptors coupled to catecholamine-sensitive adenylate cyclase, supporting a role for catecholamines in the regulation of placental metabolism.

ß 1-Adrenergic receptors in human neuroblastoma

ß-Adrenergic receptors and adenylate cyclase activity were identified and partially characterized in 10 human neuroblastoma tumors obtained from direct biopsy, from human cell lines grown in the athymic, nude mouse or from established tumor cell lines in tissue culture. Particulate membrane fractions of eight of the tumors contained ß-adrenergic receptor sites identified by the ß-adrenergic antagonists (−)-[ 3H]dihyroalprenolol ((−)-[ 3H]DHA) and (−)-[ 125I]iodohydroxybenzylphindolol ((−)-[ 125I]HYP). Specific binding of high affinity sites (B max) varied greatly among the tumors ranging from non-detectable concentrations to approximately 130 fmol/mg of crude membrane protein. Analysis of the saturation experiments with (−)-[ 3H]DHA resulted in curvilinear Scatchard plots in the tumors, suggesting the presence of multiple classes of sites and there was no evidence of cooperativity in dissociation experiments with (−)-[ 3H]DHA. The high affinity class of sites were inhibited by catecholamines, in contrast to the ineffectiveness of catecholamines in inhibiting (−)-[ 3H]DHA binding at the low affinity class of sites. TheK D of the high affinity site was approximately 1–2 nM for (−)-[ 3H]DHA and was similar in all the tumors studied. A single class of sites was demonstrated with (−)-[ 125I]HYP, in several of the tumors studied,K D approximately 120 pM, and the number of sites determined with (—)-[ 125I]HYP was identical to the number of high affinity sites determined with (−)-[ 3H]DHA. The properties of the receptor were characterized in several tumor lines: SK-N-MC tumor line contained both a ß-adrenergic receptor site and catecholamine sensitive adenylate cyclase activity. Agonists competed for the ß-adrenergic sites in the order of potency (−)-isoproterenol > (−)-norepinephrine = (−)-epinephrine > > (−)-dopamine characteristic of a ß-adrenergic receptor subtype. The affinity of the receptor for the selective agents, metropol (ß 1-selective antagonist) and zinterol (ß 2-selective agonist) was typical of a homogeneous ß 1-adrenergic site. Guanine nucleotides, guanine triphosphate and guanyl-5′-yl-imidophosphate, decreased catecholamine affinity for the receptor site in several tumors studied and enhanced maximal catecholamine stimulated adenylate cyclase activity in the sensitive cell line. Adenylate cyclase activity in particulate fractions of most of the neuroblastoma tumors was responsive to prostaglandins (PGE 1), guanine nucleotide and NaF. While most tumors contained ß-adrenergic receptor sites only SK-N-MC was clearly catecholamine responsive as assessed by catecholamine-sensitive adenylate cyclase activity. Human neuroblastomas, a homogenous tissue of neural crest origin, contained ß-adrenergic receptor sites which varied greatly in number (B max) and in their ability to stimulate adenylate cyclase activity. Guanine nucleotides decreased agonist affinity for the receptor in the lines tested and enhanced prostaglandin and catecholamine sensitive activity in responsive tumor lines.

Catecholamine-induced alteration in sedimentation behavior of membrane bound beta-adrenergic receptors.

Incubation of astrocytoma cells with catecholamines results in a decrease in catecholamine-stimulated adenylate cyclase activity and a concomitant alteration in the sedimentation properties of particulate beta-adrenergic receptors. The altered receptors exhibit agonist binding properties similar to those of receptors that are "uncoupled" from adenylate cyclase.

Use of cell fusion techniques to probe the mechanism of catecholamine-induced desensitization of adenylate cyclase in frog erythrocytes

The catecholamine-sensitive adenylate cyclase system appears to be comprised of at least three components; the beta-adrenergic receptor (R component), the catalytic unit of adenylate cyclase (C component) and a nucleotide regulatory protein (N component), responsible for mediating the effects of guanine nucleotides on the system. Cell fusion techniques were used to investigate the role of these three components in the process of homologous desensitization in the frog erythrocyte. Dicyclohexylcarbodiimide (DCCD) was used to inhibit β-receptor function in one population of frog erythrocytes, whilst phenyl glyoxal was employed to inactivate the N and C components in a second population of frog erythrocytes. Using Sendai virus to fuse the two types of modified cell, heterologous β-adrenergic receptor-adenylate cyclase systems were constructed which contained components from each cell type. When beta receptors from cells previously desensitized to catecholamines were coupled to N-C components derived from fresh erythrocytes, the resulting hybrid exhibited a densitized response to isoproterenol. By contrast, when β-adrenergic receptors from fresh cells were coupled to N-C components derived from desensitized erythrocytes, no decreased responsiveness to isoproterenol was apparent in the hybrid. That this resensitization was the result of the addition of fresh β-adrenergic receptors was demonstrated in a control experiment. Frog erythrocytes were desensitized simultaneously to catecholamines and prostaglandin E 1 and modified with DCCD which inactivates the β-adrenergic receptor but not the prostaglandin receptor. When fresh β-adrenergic receptors were supplied by cell fusion to these doubly desensitized erythrocytes, only the β-adrenergic response was restored to control levels. The response to prostaglandin remained desensitized in the hybrids, indicating that the observed resensitization of catecholamine-stimulated adenylate cyclase activity was specific and was due to the addition of fresh β-adrenergic receptors. These data suggest that in the frog erythrocyte, homologous desensitization is primarily the result of receptor-related alterations.