Donald E. Gammon

The Influence of Hyperthyroidism and Hypothyroidism on the β-Adrenergic Responsiveness of the Turkey Erythrocyte

The mechanisms responsible for altered adrenergic tone in hyperthyroidism and hypothyroidism are not fully understood. To investigate these mechanisms, the beta-adrenergic receptor-cyclic AMP complex of the turkey erythrocyte was studied among groups of normal, hyperthyroid, and hypothyroid turkeys. In erythrocytes obtained from hypothyroid turkeys, there were fewer beta-adrenergic receptors than in normal cells as determined by the specific binding of [(125)I]iodohydroxybenzylpindolol, as well as associated decreases both in catecholamine-responsive adenylate cyclase activity and in cellular cyclic AMP content. In contrast, erythrocytes obtained from hyperthyroid turkeys contained the same number of beta-receptors and had the same catecholamine-responsive adenylate cyclase activity as cells from normal birds. Other characteristics of the beta-receptors in cells from hyperthyroid birds were indistinguishable from those present in normal erythrocytes. However, within the range of circulating catecholamine concentrations, 5-50 nM, the erythrocytes of the hyperthyroid turkeys generated substantially more cyclic AMP after exposure to isoproterenol than did normal cells. These results suggest that thyroid hormone affects beta-receptor-cyclic AMP interrelationships in the turkey erythrocyte by two distinct mechanisms: (a) In hypothyroidism, both beta-receptors and catecholamine-dependent cyclic AMP formation are coordinately decreased; (b) in hyperthyroidism, beta-receptors are unchanged but there is an amplification of the hormonal signal so that occupation of a given number of receptors at physiological concentrations of catecholamines leads to increased levels of cyclic AMP.

Structure-binding—Activity analysis of beta-adrenergic amines—II: Binding to the beta receptor and inhibition of adenylate cyclase☆

Abstract Over fifty catecholamines and related analogues were tested for their ability to bind to the beta-adrenergic receptor and to activate adenylate cyclase in membranes of the turkey erythrocyte. Using [ 125 I] hydroxybenzylpindolol, a radioligand that has successfully been used to detect betaadrenergic receptors, binding to erythrocyte membranes was highly specific and showed strict selectivity for (−) antipodes. Over a range of four log orders, the affinity of a compound for the beta receptor correlated significantly with its ability to activate adenylate cyclase. Potency both for specific binding and adenylate cyclase activation was related to the size of the substituent on the secondary ethanolamine and to the configuration of the aromatic group. Full intrinsic activity required the presence of the catechol and beta-hydroxyl groups. These results confirm that [ 125 I] hydroxybenzylpindolol is a direct probe for the detection of beta-adrenergic receptors and further delineate the structural requirements for occupation and activation of the beta-adrenergic receptor.

A radioreceptor assay for propranolol.

A radioreceptor assay for the measurement of propranolol levels in human subjects is described. The assay is based upon competition between the radiolabeled β‐adrenergic inhibitor [125I]iodohydroxybenzylpindolol and propranolol for specific beta receptor sites in turkey erythrocyte plasma membranes. The radioreceptor assay is extremely sensitive, specific, and technically adaptable to processing clinical samples. Propranolol in serum is quantitatively extracted into ethyl acetate and, after the organic phase is reduced to dryness, reconstituted in aqueous assay buffer. Propranolol levels as low as 0.25 to 0.5 ng/ml can be detected and maximal sensitivity occurs at a concentration of 2.4 ± 0.2 ng/ml. The assay demonstrates specificity for biologically active metabolites of propranolol as well and therefore, with modifications, has the potential of providing a composite index of circulating β‐adrenergic inhibitory activity. Nonactive propranolol metabolites, d‐isomers, and the catecholamines do not significantly interfere in the assay. Thirty clinical samples containing a wide spectrum of propranolol concentrations were analyzed by radioreceptor assay and then compared with values obtained by high‐pressure liquid chromatography (HPLC) or by radioimmunoassay. The coefficients of correlation, r, between the radioreceptor assay and these other two methods were +0.91 and +0.85, respectively (p < 0.01). These observations establish the characteristics and the clinical feasibility of the radioreceptor assay for propranolol and suggest that it will be useful in further elucidating the kinetics of propranolol in human subjects.

Regulation of catecholamine-responsive adenylate cyclase activity in rat reticulocyte membranes by endogenous factors General characteristics and resolution into protein and nucleotide components

A 100 000 X g soluble, supernatant fraction obtained from the hemolysate of rat reticulocytes was studied for its effect upon catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. The supernatant material, devoid of adenylate cyclase activity itself, amplified isoproterenol-dependent activity in responsive membranes and was an essential requirement for the expression of hormone sensitivity in membranes rendered unresponsive to isoproterenol alone. The increment in catecholamine-associated activity conferred upon reticulocyte membranes by the supernatant material was beta-adrenergic because it did not affect basal or fluoride-related activity and was completely inhibited by propranolol. Guanine nucleotides were present in the supernatant but could account for only a fraction of the total activity because the supernatant was able to cause greater stimulation than maximal concentrations of GTP and when specified concentrations of exogenous GTP were compared with equivalent nucleotide concentrations in the supernatant, the supernatant always led to greater activity. The supernatant was resolved into protein-and nucleotide-containing components by ion-exchange chromatography. Each component was approximately one-half as active in amplifying catecholamine-dependent adenylate cyclase as the unresolved, crude supernatant material. The activity eluted in the first peak of the DEAE chromatogram was resistant to alkaline phosphatase, sensitive to trypsin, not dialyzable and contained no detectable concentrations of GTP or GDP. In contrast, the activity eluted the second peak of the DEAE chromatogram was sensitive to alkaline phophatase, resistant to trypsin, completely dialyzable and contained both GTP (30 microM) and GDP (10 microM) in significant concentrations. Neither the crude supernatant nor its two active components affected the binding of [125I]-iodohydroxybenzylpindolol to reticulocyte membranes. These observations establish in rat reticulocytes the presence of protein and guanine nucleotide constituents which have independent influences upon the catecholamine-responsive adenylate cyclase of reticulocyte membranes.

A radioreceptor assay for propranolol and 4-hydroxypropranolol.

A radioreceptor assay for the measurement of propranolol and 4‐hydroxypropranolol levels in plasma is described. Maximum sensitivity for propranolol was 1.2 ± 0.15 ng/ml and for 4‐hydroxypropranolol 4.2 ± 0.4 ng/ml. Interassay and intra‐assay variations for both were under 10%. Modifications in the radioreceptor assay permitted the measurement of total β‐adrenergic blocking activity and the separate contributions of parent drug and metabolite. When 4‐hydroxypropranolol was stabilized, a composite level of total β‐adrenergic blocking activity in plasma was obtained. When the 4‐hydroxy metabolite was oxidized, only the stable parent drug was detected. The difference in values between measurements made under these conditions was equivalent to the amount of 4‐hydroxypropranolol in the sample. The radioreceptor assay was also used to measure the amount of free propranolol and 4‐hydroxypropranolol. Under identical experimental conditions, more 4‐hydroxypropranolol than propranolol circulated in the free form. These observations establish the feasibility of adapting the radioreceptor assay for propranolol to the measurement of total β‐adrenergic blocking activity and its components in plasma as well as to the measurement of free drug and metabolite levels.

The effect of age on beta-adrenergic receptors and adenylate cyclase activity in rat erythrocytes

Abstract Beta-adrenergic receptors and catecholamine-sensitive adenylate cyclase activity were studied in erythrocytes obtained from rats 6 weeks, 6 months, and 15 months of age. Intact erythrocytes from 6 week old rats contained significantly more beta receptors (411 ± 31 sites/cell) than 6 month (328 ± 21) or 15 month old rats (335 ± 16), as determined by binding of [ 125 I] iodohydroxybenzylpindolol. Erythrocytes from 6 week old rats also contained significantly greater isoproterenol-sensitive adenylate cyclase activity (95.0 ± 9.4pmoles/10 9 cells) than erythrocytes from 6 month (27.9 ± 3.3) or 15 month old rats (23.7 ± 3.6). The erythrocyte population of 6 week old rats was bigger (mean corpuscular volume = 62 ± 2 μ 3/cell ) than the older rat erythrocytes (47 ± 1 μ 3 and 48 ± 1 μ 3 ). When the data were expressed relative to a unit of cell volume, there was no difference in the density of beta receptors among all three populations but a progressive and significant fall in hormone-sensitive adenylate cyclase activity. In the rat erythrocyte, the age-related loss of adenylate cyclase activity is not accompanied by changes in β-receptor density.

Guanine nucleotide-induced shift in binding affinity for β-adrenergic agonists in rat reticulocyte and turkey erythrocyte membranes

Abstract Guanosine triphosphate (GTP) is important for the expression of catecholamine-dependent adenylate cyclase activity in a wide variety of membrane preparations, including those obtained from rat reticulocytes and turkey erythrocytes. Another generally recognized effect of GTP—to decrease the binding affinity of β-adrenergic agonists for β receptor sites—has not been demonstrable, until recently, in these two membrane preparations. The present study was designed to evaluate possible guanine nucleotide effects on agonist binding under clearly defined experimental conditions. In the absence of magnesium and EDTA, the approximate binding constant K D (app) , for isoproterenol did not change significantly in the presence of the GTP analogue, guanyl-5′-yl-imidodiphosphate [Gpp(NH)p], for either rat reticulocytes (14.0 ± 3.6 vs 19.5 ± 1.8 μ M; P > 0.10) or turkey erythrocytes (2.66 ± 0.92 vs 2.33 ± 0.07 μ M; P > 0.10). When magnesium (10 mM) and EDTA (1 mM) were both present, the K D (app) for isoproterenol improved 23-fold in the rat reticulocyte to 0.62 ± 0.1 μ M (P μ M (P K D (app) for isoproterenol occurring at a magnesium concentration of 0.1 mM. When binding experiments were conducted in the presence of magnesium and EDTA, Gpp(NH)p caused a marked decrease in agonist affinity in membranes of rat reticulocytes (0.62 ± 0.10 to 9.3 ± 3.3 μ M; P μ M; P

A cellular activator of catecholamine-sensitive adenylate cyclase in rat reticulocytes and erythrocytes: Changes during reticulocyte development and effects on the β receptor

Abstract Rat reticulocytes contain an isoproterenol-sensitive adenylate cyclase activity which is lost with maturation to erythrocytes despite no change in the density of β-adrenergic receptors. To explore this observation, a cytosol factor, previously shown to be important in the expression of catecholamine-sensitive adenylate cyclase in the reticulocyte, was compared to a cytosol factor obtained in a similar manner from mature erythrocytes. The cytosol factor from reticulocytes augmented isoproterenol-responsive adenylate cyclase activity in reticulocyte and erythrocyte membranes half-maximally at 0.7 ± 0.1 (SEM) and 1.1 ± 0.3 μg/ml, respectively. These concentrations of reticulocyte-derived cytosol factor were significantly lower ( P P m ; P

Organ culture of human parathyroid glands: Effects of catecholamines☆

Normal and adenomatous human parathyroid glands were studied in organ culture to determine basal secretory rates of cyclic AMP and parathyroid hormone as well as sensitivity to β-adrenergic catecholamines. Basal cyclic AMP secretion was relatively constant over 4 weeks but parathyroid hormone secretion declined. Both normal and adenomatous tissue were stimulated to secrete cyclic AMP and parathyroid hormone in the presence of the β agonist, isoproterenol. Half-maximal stimulation for adenomas occurred at 1 μM. Propranolol, a β-adrenergic inhibitor, completely prevented the response, with half-maximal inhibition occurring at 0.1 μM. Under basal conditions, normal explants secreted significantly more cyclic AMP and parathyroid hormone than adenomas. The results indicate that both normal and adenomatous human parathyroid glands can be maintained in organ culture for up to 4 weeks and that β-adrenergic catecholamines can stimulate the secretion of cyclic AMP and parathyroid hormone.