Β-adrenergic Catecholamines Research Articles

A stress response pathway regulates DNA damage through β2-adrenoreceptors and β-arrestin-1.

The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates β(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated β(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of β-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which β-adrenergic catecholamines, acting through both Gs-PKA and β-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, β-arrestin-1 (ARRB1), activated via β(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10-6 mol·l-1) induces a 30- to 40-fold activation of Na+/H+ exchange (the ethylisopropylamiloride-inhibited component of the 22Na influx) and a fourfold stimulation of the Na+, K+ pump (ouabain-inhibited component of 86Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (β-phorbol 12-myristate, 13-acetate) increases Na+/H+ exchange by 10 times and decreases the Na+, K+ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na+, K+ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na+/H+ exchange-independent mechanism of the Na+, K+ pump regulation by β-adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na+/H+ exchange, whereas hypotonic swelling induces an increase in the rate of 86Rb+ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH0 recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na+/H+ exchange, Na+, K+ pump and a non-identified system providing for K+ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.

Triiodothyronine causes rapid reversal of α 1/cyclic adenosine monophosphate synergism on brown adipocyte respiration and type II deiodinase activity

Previous studies have shown that thyroid status affects the response of brown adipose tissue (BAT) to the sympathetic nervous system. For example, hypothyroidism is associated with the development of a marked synergism between α 1- and β-adrenergic pathways to stimulate type II iodothyronine 5′-deiodinase activity. Hypothyroidism also attenuates the respiratory response (thermogenesis) of isolated brown adipocytes to norepinephrine. To explore the interactions of the sympathetic nervous system and thyroid status in these cells, we compared the thermogenic and 5′-deiodinase responses to adrenergic agonists in isolated brown adipocytes from hypothyroid rats during treatment with 3,5,3′-triiodothyronine (T 3). The fivefold synergism of α 1- and β-adrenergic catecholamines to increase the deiodinase activity was progressively reduced, reaching a control euthyroid value of unity after 5 days of T 3 treatment. Hypothyroidism reduced both the O 2max (twofold to threefold) and increased the concentration of agonist required for 50% stimulation (10-fold) for both norepinephrine and forskolin. In hypothyroid cells, there was a twofold synergism between the α 1-agonist cirazoline and forskolin to increase respiration, which was blocked by prazosin and reproduced by the calcium ionophore, A23187. This synergistic effect of the α 1-agonist was lost within 2 days of T 3 administration. These studies identify a second Ca 2+-dependent intraadrenergic synergism, which functions to ameliorate the reduced cyclic adenosine monophosphate (cAMP) responsiveness of the hypothyroid brown adipocyte.

Cholinergic regulation of impulse frequency in peripheral nerve

Previous studies have demonstrated the existence of receptors for γ-aminobutyric acid (GABA), β-adrenergic catecholamines and acetylcholine in vertebrate peripheral nerve, and provided functional correlates for activation of both GABA and β-adrenergic receptors. The present studies show that a cholinergic receptor present on the nerve can also modify impulse pattern. In frog sciatic nerve, both carbamylcholine and dibutyryl cyclic GMP increased the amplitude of the response to the second stimulus of a train at very short interstimulus intervals. The effect of carbamylcholine was blocked by 4-aminopyridine. The results are consistent with the hypothesis that cholinergic agonists mediate an increase in endogenous cyclic guanosine monophosphate (cGMP), which increases the ability of the nerve to follow closely spaced stimuli by inhibiting potassium channels.

Properties And Modulation of Cardiac Calcium Channels

Voltage-dependent calcium channels are widely distributed in excitable membranes and are involved in the regulation of many cellular functions. These channels can be modulated by neurotransmitters and drugs. There is one particular type of calcium channel in cardiac cells (L-type) whose gating is affected in different ways by beta-adrenoceptor and 1,4-dihydropyridine agonists. We have analysed single calcium channel currents (i) in myocytes from rat hearts in the absence and presence of isoproterenol or 8-bromo-cAMP. We have found that both compounds have similar effects on calcium channel properties. They increase the overall open state probability (po) of individual calcium channels while i remains unaffected. Analysis of the gating kinetics of calcium channels showed: a slight increase in the mean open times of calcium channels, a reduction in time intervals between bursts of channel openings, an increase in burst length and a prominent reduction in failures of calcium channels to open upon depolarization. These kinetic changes caused by isoproterenol and 8-bromo-cAMP can account for the increase in po. Since the macroscopic calcium current, ICa, can be described by ICa = N X po X i, the increase in po accounts for the well-known increase in ICa by beta-adrenergic catecholamines. Cyclic AMP-dependent phosphorylation of calcium channels is a likely metabolic step involved in this modulation. Another class of drug that modulates calcium channel gating is the 1,4-dihydropyridines which can either enhance or reduce ICa, either by prolonging the open state of the channels or by facilitating the inactivated state. Both effects depend strongly on membrane potential and are independent of cyclic AMP-dependent phosphorylation reactions.

Alteration of the pattern of β-adrenergic desensitization in cultured L 6E 9 muscle cells infected with Trypanosoma cruzi

L 6E 9 myoblasts infected with Trypanosoma cruzi undergo desensitization to β-adrenergic catecholamines in a manner distinct from uninfected control myoblasts. Following incubation of intact cells with isoproterenol for 2h, homogenates prepared from differentiated, high density uninfected L 6E 9 cells retain isoproterenol-dependent adenylate cyclase activity. In addition, previous exposure to isoproterenol is accompanied by a decrease in the number of β-adrenergic receptors. Homogenates of high density L 6E 9 cells infected with T. cruzi retain their adenylate cyclase responsivity to isoproterenol but demonstrate a marked decrease in β-adrenergic receptors. Following desensitization infected cell homogenates lose their responsiveness to isoproterenol and demonstrate a more marked decrease in β-receptors. There does not appear to be any effect of T. cruzi infection on affinity of β-adrenergic agonists for the β-receptor or on changes in agonist affinity associated with desensitization. Infection of low density undifferentiated cells results in no apparent change in adenylate cyclase activity or in β-receptors. Their behavior in the setting of desensitization — decreased whole cell cyclic AMP, decreased adenylate cyclase, unchanged β-receptors — is also not affected by infection. The pattern of desensitization to β-adrenergic agonists in high density infected cells shares several properties with the pattern of desensitization in low density uninfected cells, suggesting that infection may be associated with part of the more primitive cellular response pattern.

Desensitization of cultured muscle cells to isoproterenol

We have studied the desensitization of the muscle cell line L 6E 9 to isoproterenol as a function of cell density in both whole cells and homogenates derived from them. Undifferentiated (low density) and differentiated (high density) cells both respond to isoproterenol initially with a rapid elevation of cellular cyclic AMP. High density cells require 8 h (versus 4 h for low density cells) of continuous exposure to isoproterenol before cyclic AMP levels are reduced to control values. This difference between high and low density cells would appear to be due to greater phosphodiesterase activity in low density cells. When either low or high density cells are continuously exposed to isoproterenol for 2 h, washed and reexposed to fresh isoproterenol, cyclic AMP levels can no longer be increased above corresponding controls. In this respect, desensitization to isoproterenol in low and high density cultures proceeds with a similar time course. However, membranes prepared from intact desensitized low and high density cells show differences from each other both in isoproterenol-dependent adenylate cyclase activity and in β-adrenergic receptors. Desensitized low density cells lose, whereas desensitized high density cells retain, isoproterenol-dependent adenylate activity. On the other hand, the number of β-receptors, as detected by [ 125I]iodocyanopindolol, is unchanged in low density cells but decreased in high density cells. Furthermore, the affinity of isoproterenol for β-receptors is decreased to a greater extent in high density than in low density cells. The results indicate that desensitization of β-adrenergic catecholamines involves several different loci in the hormonal machinery of the cell and that the importance of these loci varies as a function of cellular differentiation.

Prostacyclin and β-Adrenergic Catecholamines Inhibit Arachidonate Release and PGI2 Synthesis by Vascular Endothelium

AbstractWe have investigated the mechanism by which cyclic AMP inhibits PGI2 synthesis in cultured bovine aortic endothelial cells. Inhibition of cyclic AMP phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX) blocks calcium ionophore-induced PGI2 production by 62%. The addition of 3 mM dibutyryl cyclic AMP, along with IBMX, increases the inhibition to 96%. Release of 3H-arachidonate from membrane phospholipids was inhibited 21% by dibutyryl cyclic AMP, 48% by IBMX, and 76% by isoproterenol plus IBMX. Inhibition by isoproterenol was reversed by 10 μM propranolol. Release of 3H-arachidonate was also reduced 75% by a combination of 10 μM PGI2 and 3 mM IBMX. We conclude that hormones like isoproterenol and PGI2 may regulate endothelial cell PGI2 biosynthesis by increasing intracellular cyclic AMP, which then inhibits release of endogenous arachidonate from membrane phospholipids.

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 μ M. Propranolol, a β-adrenergic inhibitor, completely prevented the response, with half-maximal inhibition occurring at 0.1 μ M. 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.

Copurification of beta-adrenergic catecholamine receptors and hormone-sensitive adenylate cyclase with uterine smooth muscle plasma membranes.

AbstractA putative plasma membrane fraction isolated from rat uterine smooth muscle was substantially enriched in both β-adrenergic catecholamine receptors as detected by radioligand binding methods and adenylate cyclase. Receptors for adrenergic catecholamines as well as series E prostaglandins were functionally coupled to the enzyme in the purified membranes by a GTP-requiring process. Localization of receptors and enzymes to the same subcellular compartment suggest that in myometrium, as in other target tissues, β-adrenergic catecholamines and perhaps series E prostaglandins exert their effects by regulating cAMP production.

Increasing number of β-adrenergic receptors in intact, differentiating muscle cells

In order to assess changes in β-adrenergic receptors that occur with differentiation of myogenic cells, a method was developed to determine the number of β receptor sites in intact cells in situ . Conditions of the binding assay were established to maximize specific binding of [ 125I]iodohydroxybenzylpindolol to cells and to insure that neither trypsin nor EDTA used for passaging purposes interfered with the assessment of binding sites. With differentiation, a 3-fold increase in β-adrenergic receptors in intact cells was observed. Characteristics of the β receptors when compared to cells prior to differentiation were otherwise unchanged with similar association and dissociation kinetics and binding affinity. These results confirm previous observations in broken cell preparations, provide a reasonable estimate for the number of β receptors in these cells, both before and after differentiation, and provide support for the possibility that the β-adrenergic catecholamines are important in the differentiation of myogenic cells.

Evidence of a new, preferentially Mg-carrying, transport system besides the fast Na and the slow Ca channels in the excited myocardial sarcolemma membrane

MgCl 2 added to a K-rich (19 m m Ca-deficient (0.4 m m) Tyrode solution in a suitably high concentration (14 to 34 m m Mg) maintains or restores electric excitability, overshoot and propagated action potentials. The experiments reveal the true existence of a special transport system for the Mg entry into the ventricular myocardium that can be distinguished from both the fast Na and the slow Ca channels by means of certain drugs and appropriate electrophysiological techniques. The transmembrane transport systems for Na, Mg, and Ca ions differ with respect to their sensitivities to graded membrane depolarizations. The Na channel is most easily inactivated at a reduced level of membrane potential, whereas the Ca channel is most resistant. The Mg channel has an intermediate position. Ca-mediated action potentials are selectively blocked by specific Ca-antagonists such as verapamil, D 600, nifedipine or diltiazem. However, Mg-carried action potentials are refractory to these inhibitors. Tetrodotoxin and lidocaine are capable of suppressing both the transmembrane Na and Mg influxes without affecting the slow Ca channel. β-Adrenergic catecholamines influence the myocardial Mg and Ca currents in an exactly opposite sense: β-adrenergic substances largely promote myocardial Ca uptake and Ca-dependent action potentials whereas they totally block the transmembrane Mg fluxes into the partially depolarized myocardial fibres and, thereby, inhibit every Mg-dependent biolectric activity. Hence the usual “two-channel concept” that only allowed for a fast Na and a slow Ca inward current, but ignored the existence of a third transmembrane transport system that preferentially carries Mg ions into the ventricular myocardium, has to be considered as an oversimplification. It is suggested that the reciprocal effects of β-adrenergic catecholamines on the inward fluxes of Ca and Mg ions serve to maximize the contractile responses to β-receptor stimulation. The intensification by β-adrenergic catecholamines of the Ca influx simultaneously with a restriction of the transmembrane supply of Ca-antagonistic Mg ions will activate Ca-dependent excitation-contraction coupling of the ventricular myocardium in the most effective manner.

Decreased beta-adrenergic receptors on polymorphonuclear leukocytes after adrenergic therapy.

β-ADRENERGIC catecholamines, which modulate function of a variety of tissues, are assumed to act by initially binding to receptors on target-cell plasma membranes, thereby activating adenylate cyclase and stimulating generation of adenosine 3′:5′-monophosphate (cyclic AMP). In addition to activating adenylate cyclase, β-adrenergic agonists commonly have a second action — regulation of responsiveness of the target cells. Thus, continued treatment with such agonists often results in decreased cellular response. This self-regulation of target-cell responsiveness, variously termed refractoriness, desensitization and down-regulation, has been observed with catecholamines, as well as with a number of other hormonal agonists.1 2 3 For catecholamines, studies in several experimental . . .

Dissociation of beta-adrenergic receptors from hormone responsiveness during maturation of the rat reticulocyte

Intact rat erythrocytes and reticulocytes have been studied in relation to their concentration of β-adrenergic receptors and their responsiveness to β-adrenergic catecholamines. Characteristics of the β-receptor, as determined by binding of 125I-labelled hydroxybenzylpindolol, were compared among control erythrocytes and reticulocytes. The dissociation constant ( K d=0.1−0.2 nM), association and dissociation kinetics, and stereospecificity for (−)-isomers of agonists and antagonists were similar in both cell types. The reticulocyte population contained four times more receptors per cell than the control erythrocytes. However, reticulocytes were 25 times more responsive than control cells to isoproterenol, as measured by the formation of cyclic AMP. After peak reticulocytosis, cells rapidly lost 95% of their maximum hormone responsiveness, but β-receptors declined much more slowly. The 4-fold decrease in β-receptors was associated with a 4-fold decrease in cell volume as the reticulocytes matured. The density of β-receptors was unchanged. However, responsiveness to isoproterenol in the reticulocytes when expressed on the basis of cell volume was still nine times greater than the control cells. Thus, maturation of reticulocytes is associated with an uncoupling of persistent β-receptors from catecholamine responsiveness.

Adenylate cyclase system of human skeletal muscle: Characteristics of catecholamine stimulation and nucleotide regulation

Human skeletal muscle plasmalemmal adenylate cyclase activation by catecholamines and guanyl nucleotides was investigated. The enzyme was selectively stimulated by different β-adrenergic catecholamines. Their order of potency was isoproterenol > epinephrine > norepinephrine. Phenylephrine, an α-adrenergic agonist, showed no effect. Stimulation of enzyme by β-agonists was stereospecific as (−)-isomers of isoproterenol and epinephrine were at least two orders of magnitude more potent than their corresponding (+)-isomers. The β-adrenergic blocking agents, propranolol and alprenolol, completely abolished the stimulation of adenylate cyclase by (−)-isoproterenol. Inhibition by β-blockers was also stereospecific, (−)-isomers being more effective inhibitors than their corresponding (+)-isomers. This selectivity and stereospecificity suggests that the human plasmalemmal adenylate cyclase is closely associated with the β-adrenergic receptors. Purine nucleotides, particularly GTP and to a lesser extent ITP, further enhanced the catecholamine-sensitive, but not the basal, adenylate cyclase. Pyrimidine nucleotides, UTP and CTP, were without any effect on the enzyme activity. Of the guanosine compounds tested only GTP, and to a lesser extent GDP, enhanced the enzyme activity in the presence of (−)-isoproterenol, while guanosine, GMP and cyclic GMP were ineffective. 5′-Guanylyl imidodiphosphate (GMP-PNP), a non-phosphorylating analog of GTP, was a more profound activator of catecholamine-sensitive adenylate cyclase than GTP. In contrast to GTP, GMP-PNP stimulated the basal enzyme activity. The plasmalemmal adenylate cyclase showed greater affinity towards GTP ( K m 3·10 −7 M) than towards GMP-PNP ( K m 2·10 −6 M). Profound augmentation by GTP and GMP-PNP of plasmalemmal adenylate cyclase in the presence of (−)-isoproterenol appeared to be a function of the β-adrenergic receptor because propranolol completely abolished their effects; GTP competitively inhibited that marked augmentation caused by GMP-PNP, suggesting that both nucleotides interact at similar sites on the enzyme system. Kinetic studies revealed that the predominant effect of guanyl nucleotides was to enhance the catalytic activity of the enzyme without altering its affinity for ATP, (−)-isoproterenol or Mg 2+. It is concluded that the human plasmalemmal adenylate cyclase is closely associated with the β-adrenergic receptor and that the guanyl nucleotides are potent regulators of the plasmalemmal enzyme.

Agonist-induced increase in apparent β-adrenergic receptor size

The properties of digitonin-solubilized beta-adrenergic receptors from frog erythrocyte membranes were studied by gel exclusion chromatography on AcA 34 Ultragel. beta-Adrenergic receptor binding activity in these membranes can be identified by both an agonist ligand, [(3)H]hydroxybenzylisoproterenol, and the antagonist ligands, [(3)H]dihydroalprenolol and (125)I-labeled hydroxybenzylpindolol. Occupancy of the beta-adrenergic receptors with the [(3)H]hydroxybenzylisoproterenol agonist prior to their solubilization from the membrane leads to an increase in apparent receptor size. Alterations in the molecular size of the receptor cannot be mimicked by occupancy of the binding site with the antagonist ligands. Exposure of frog erythrocyte membranes to [(3)H]hydroxybenzylisoproterenol agonist in the presence of 10 muM Gpp(NH)(p), a guanyl nucleotide analog that exerts multiple regulatory effects on the catecholamine-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] system, results in the elution of the [(3)H]hydroxybenzylisoproterenol radioligand in both the region characteristic of the agonist-receptor complex and the region characteristic of the antagonist-receptor complex. The precise molecular interactions responsible for the agonist-induced increase in apparent beta-adrenergic receptor size are still unresolved. However, the low concentrations of agonist that are capable of altering apparent receptor size and the sensitivity of this effect to guanyl nucleotides suggest that these phenomena may be intimately involved in eliciting the physiological effects of beta-adrenergic catecholamines at the molecular level.

Regulation of Adenylate Cyclase Coupled β-Adrenergic Receptors by β-Adrenergic Catecholamines

Injection of frogs with beta-adrenergic catecholamines produced a selective desensitization (loss of responsiveness) of the erythrocyte membrane adenylate cylase to subsequent stimulation in vitro by isoproterenol. Basal, prostaglandin E1- and fluoride-sensitive enzyme activities were unaffected. A 77% (p less than 0.001) decline in isoproterenol-responsive enzyme activity in the cells from the treated animals was observed with no change in the Km for isoproterenol stimulation of the enzyme (concentration causing 1/2 maximal enzyme activation). The decrease in catecholamine-sensitive adenylate cyclase was accompanied by a parallel 68% (p less than 0.001) fall in the apparent number of beta-adrenergic receptors in the erythrocyte membranes, assessed by (-) (3H)alprenolol binding studies. There was no change in the affinity of the receptor binding sites. The catecholamine-induced desensitization and fall in the beta-adrenergic receptor number were both concentration and time-dependent and displayed beta-adrenergic specificity. Isoproterenol was more potent in desensitizing cells and in lowering the receptor number than was norepinephrine. The beta-adrenergic antagonist propranolol, but not the alpha-adrenergic antagonist phentolamine, blocked the desensitizing effects of isoproterenol. Propranolol itself, however, did not cause desensitization. Cells became resensitized to the stimulatory effects of catecholamines, in association with a return in beta-receptor number, when propranolol was injected into previously desensitized animals. The changes in receptor number in membranes from desensitized and resensitized animals were also reflected in soluble receptor preparations. The protein synthesis inhibitor cycloheximide did not affect either desensitization, resensitization, or the changes in receptor number which accompanied the changes in adenylate cyclase sensitivity to catecholamines. These findings suggest that the chronic occupancy of beta-adrenergic receptors by beta-adrenergic agonists (but not antagonists) decreases the number of functional beta-adrenergic receptor binding sites and, hence, lowers the responsiveness of adenylate cylase to catecholamine stimulation. The lack of effort of cycloheximide on these regulatory effects suggests that "inactivation" and subsequent "reactivation" of the receptors, rather than changes in receptor turnover, are involved.

Stimulation of sodium transport in turkey erythrocytes by cyclic 3',5'-AMP.

At relatively low concentrations the β-adrenergic blocking agent, propranolol, inhibits competitively isoproterenol-stimulated sodium transport in intact turkey erythrocytes. At higher concentrations propranolol alone inhibits sodium transport; however, this effect is probably not mediated by the adenylate cyclase system. When propranolol was added after turkey erythrocytes had been stimulated by isoproterenol, there was a progressive decrease in sodium influx and an associated decrease in cellular cyclic 3',5'-AMP. These results, plus the observation that addition of cyclic 3',5'-AMP to the medium stimulated sodium transport, offer further support for the conclusion that stimulation of sodium transport in turkey erythrocytes by β-adrenergic catecholamines is mediated by activation of adenylate cyclase and consequent accumulation of cellular cyclic 3'5'-AMP. Our results also illustrate that the relation between total cellular cyclic 3'5'-AMP and sodium transport changes over time suggesting a developing refractoriness to or acompartmentalization of cellular cyclic 3',5'-AMP. Neither theophylline nor caffeine altered sodium transport or potentiated the effect of submaximal concentrations of isoproterenol; however, both methylxanthines inhibited the degradation of extracellular cyclic 3',5'-AMP and potentiated the stimulation of sodium transport by extracellular cyclic nucleotide. These results suggest that in the turkey erythrocyte phosphodiesterase may be located exclusively on the external surface of the erythrocyte membrane and acts only on extracellular cyclic nucleotides. (Endocrinology95: 499, 1974)

Surface interaction of [ 3H]norepinephrine with cultured chick embryo myocardial cells

Cultured chick embryo cardiac myoblasts specifically bind [ 3H]nonrepinephrine. The binding is rapid and reversible. Bound [ 3H]nonrepinephrine, dissociated by 1 M HCl, can be rebound to fresh cells. β-Adrenergic catecholamines were most potent in displacing [ 3H]nonrepinephrine from the cellular bindign sites. The binding reaction did not show stereospecificity. α-Adrenergic amines were much less potent. Propranolol, but no phentolamine, competed for the sites. Approximately 2.5 · 10 6 specific binding sites are present per myocardial cell. The sites appear to be present predominantly at the cell surface in that nonrepinephrine linked to agarose beads competes for th sites. Similarly, the sites were degraded by either trypsin or trypsin bound to agarose. Two different binding constants, K = 2 · 10 6 and 1 · 10 5, were observed. Proteolytic enzymes decreased binding whereas certain hospholipases led to an increase in specific binding. Divalent cations at concentrations > 1 mM diminished binding as did chelating agents.

Effect of β-Adrenergic Catecholamines on Sodium Transport in Turkey Erythrocytes

Abstract Sodium fluxes across the plasma membrane of turkey erythrocytes are 2 to 3 times greater than corresponding fluxes in human erythrocytes. Extracellular potassium stimulates fractional sodium outflux and inhibits sodium influx in turkey erythrocytes. In contrast to human erythrocytes, turkey erythrocytes showed no evidence for sodium-sodium exchange, and at all sodium concentrations studied addition of extracellular potassium inhibited sodium influx. Isoproterenol stimulated accumulation of cyclic adenosine 3':5'-monophosphate (cyclic 3':5'-AMP) and produced a 2- to 4-fold increase in sodium fluxes in turkey erythrocytes. The potency of catecholamine analogs for stimulation of sodium transport correlated directly with their potency for activation of adenylate cyclase in the turkey erythrocyte membrane. Stimulation of sodium transport by isoproterenol is reproduced by adding cyclic 3':5'-AMP to the medium and is blocked by propranolol. The increase in cyclic 3':5'-AMP in response to isoproterenol progresses unimpaired for at least 2 hours. The increase in sodium transport, however, begins to diminish within 30 min after addition of isoproterenol, and after 3 hours of incubation sodium fluxes are only slightly greater than for the control. This phenomenon presumably reflects accumulation of an inhibitor or depletion of a necessary factor for the transport system activated by intracellular cyclic 3':5'-AMP. The rapid effect described here of catecholamines on sodium transport as well as certain results with other systems controlled by hormones through cyclic 3':5'-AMP suggest that endocrine regulation of sodium transport is of general biological significance.