Mechanism of the radioprotective effect of catecholamine receptor agonists. Inclusion in the radioprotective effect of both subtypes of alpha-adrenoreceptors

alpha-Adrenoceptor agonists of both main groups, i. e. arylalkylamines and imidazolines, have a pronounced radioprotective effect. Their chemical analogs, which fail to stimulate alpha-adrenoceptors, do not protect mice. The effect of phenylephrine, adrenaline, and noradrenaline comes into play via alpha 1-adrenoceptors and that of clonidine, via alpha 2-adrenoceptors and also via alpha 1-adrenoceptors. Adrenoceptor agonists can probably manifest their radioprotective action via both subtypes of alpha-adrenoceptors. Possible intracellular mechanisms of the radioprotective action are discussed.     

Effect of hyperthermia on the radioprotective action of cysteamine and isoproterenol

Heating of mouse bone marrow cells up to 42 degrees C was shown to increase their radiosensitivity (DMF = 0.80 +/- 0.12). At this temperature, the radioprotective efficiency of cysteamine was lost completely (DMF = 0.78 +/- 0.09), and radioprotective activity of d,l-isoproterenol significantly decreased (DMF declined from 2.41 +/- 0.23 to 1.67 +/- 0.16). It is assumed that the radioprotective effect of cysteamine on mammalian cells is associated with the processes of the postirradiation DNA repair for just these processes are inhibited by heating. The mechanism of action of a beta-agonist of isoproterenol is perhaps only partially associated with DNA repair.     

Coexistence of beta 1- and beta 2-adrenoceptors in the melanophore of the goby Tridentiger obscurus

The effects of beta-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the beta-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these beta-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both alpha- and beta-adrenoceptors, and that the activation of the beta-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two beta 1-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of beta 1-receptors. By contrast, the pigment-dispersing effects of two beta 2-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the beta-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest co-existence of beta 1- and beta 2-adrenoceptors in the melanophores. The relative numbers of beta 1- and beta 2-adrenoreceptors as a percentage of the total population of beta-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the beta-agonists on the melanophores in the presence of butoxamine or metoprolol.     

Depression of the radioprotective effect of isoproterenol on mammalian cells in vitro after desensitization of the cAMP system

A short (5 min) incubation of cultured Chinese hamster fibroblasts with the specific beta-agonist isoproterenol (1 microM) leads to an increase in the intracellular content of cAMP and a decrease in radiosensitivity of the cells. Prolonged (up to 1 h) incubation induces a desensitization of the cAMP system to isoproterenol and causes a decrease both in the cAMP-stimulating and radioprotective effect of isoproterenol. There were no detectable changes in the beta-adrenoreceptor number or binding affinity of beta-receptors to the radiolabelled beta-antagonist dihydroalprenolol in desensitized cells; cAMP-phosphodiesterase activity was also the same as in intact cells. It is proposed that a 1 h incubation of the cells with isoproterenol induces the first step of desensitization, i.e. the functional "uncoupling' of beta-receptors from adenylate cyclase. Thus, the presence of beta-receptors in cells is not enough for the realization of the radioprotective potency of isoproterenol; an intact, non-desensitized, state of the cAMP system is obligatory.     

Modification of beta-adrenoceptors and adenylyl cyclase in hearts perfused with hypochlorous acid

It is now well known that the signal transduction pathway involving beta-adrenoceptors and adenylyl cyclase is altered in ischemic heart disease. Since leukocytes accumulate in the ischemic heart and produce hypochlorous acid (HOCl), we investigated the effects of HOCl upon beta-adrenoceptors and adenylyl cyclase activities by perfusing rat hearts with 0.1 mM HOCl for 10 min and isolating cardiac membranes. Marked depressions in both the density and affinity of beta1-adrenoceptors were observed, whereas no significant change in the affinity or density of beta2-adrenoceptors was seen in hearts perfused with HOCl. After treatment of hearts with HOCl, competition curves using isoproterenol, a beta-adrenoceptor agonist, revealed a decrease in the proportion of high affinity binding sites. The adenylyl cyclase activities in the absence and presence of forskolin, NaF, Gpp(NH)p, or isoproterenol were depressed in hearts perfused with HOCl; however, the stimulatory effects of these agents on adenylyl cyclase were either unaltered or augmented. The presence of methionine in the perfusion medium prevented the HOCl-induced changes in beta1-adrenoceptors and adenylyl cyclase activity. These results suggest that HOCl may produce a defect in the beta-adrenoceptor linked signal transduction mechanism by affecting both beta1-adrenoceptors and adenylyl cyclase enzyme in the myocardium.     

Beta-adrenergic receptors of the normal heart and in heart failure

The heart is often refereed to as an "beta-adrenergic organ" because beta-adrenergic agonists are powerful stimulants of cardiac contractility. Catecholamines acting through beta-adrenoceptors produce both positive inotropic and chronotropic effects in human heart. It is now generally accepted that in human heart both beta 1- and beta 2-adrenoceptors coexist. beta-Adrenergic transduction system consist of membrane-bound beta-receptors, the effector enzyme adenylyl cyclase and guanine nucleotide-binding transduction (G) proteins. Repeated long-lasting agonist stimulus evokes homologous or heterologous desensitization of transduction system. Chronic heart failure accompanies with decreased responsiveness to beta-adrenoceptor agonists and is thought to exacerbate the loss of cardiac contractility. Depending on the etiology of heart failure abnormalities of the beta-receptor-G protein-adenylyl cyclase system result from a reduced of beta 1-receptors, uncoupling of beta 1- or beta 2-receptors, alteration of G-protein function, or decreased catalytic subunit activity of adenylyl cyclase and enhanced expression of beta-adrenoceptor kinase. The model most widely used is that of circulating lymphocytes that contain a homogeneous population of beta 2-adrenoceptors. The biochemical and pharmacological properties of human lymphocyte beta 2-adrenoceptors are quite comparable to those of heart beta 2-receptors. The analysis of lymphocyte beta 2-adrenoceptor-adenylyl cyclase system can be used as a model for long-term regulation of human cardiac beta 1- and beta 2-adrenoceptors only if serial changes in response to administration of non-selective beta-adrenergic agonists or antagonists are being investigated. This review concentrates on beta-adrenoceptors in human healthy heart and in heart failure and also on lymphocyte beta 2-adrenoceptors and on the changes of these receptors properties under the influence of some cardiotropic drugs.     

The role of the hypoxic mechanism in the radioprotective effect of mezaton

The method of coupled estimation of the hypoxic and radioprotective effect has demonstrated that the hypoxic mechanism does not play an essential role in the radioprotective action of phenylephrine hydrochloride.     

Agonist-Induced Regulation of Adrenoceptor Subtypes in Cerebral Cortical Slices

Cerebral cortical slices from rat brain were incubated at 37 degrees C for 2 h in the presence of isoproterenol, noradrenaline, or adrenaline, and binding affinities and densities of adrenoceptor subtypes were subsequently examined in homogenized tissue. The density of alpha 2- and total beta-adrenoceptors was estimated using the radioligands [3H]rauwolscine and [3H]dihydroalprenolol (DHA), respectively. The percentages of beta 1- and beta 2-adrenoceptors were defined by inhibiting the binding of [3H]DHA with the beta 1-selective antagonist metoprolol. Exposure of slices to noradrenaline and adrenaline significantly decreased the maximal number of binding sites (Bmax) of alpha 2-adrenoceptors (48 and 37% respectively) without significantly affecting affinity; isoproterenol had no effect. Exposure to isoproterenol, noradrenaline, and adrenaline significantly decreased the Bmax of beta-adrenoceptors (by 60, 34, and 24%, respectively) but did not affect the affinity. Isoproterenol and adrenaline significantly decreased the density of beta 1-adrenoceptors by 75 and 24% and beta 2-adrenoceptors by 23 and 28%, respectively. Noradrenaline significantly decreased the density of beta 1-adrenoceptors by 42% without affecting the number of beta 2-adrenoceptors. These findings indicate that subtypes of adrenoceptors in rat cerebral cortex are differentially regulated by adrenergic agonists.     

Mechanism of the radioprotective effect of cysteamine

The radioprotective effect of cysteamine combined with the modification of the chromatin state by sodium butyrate has been studied using V-79 and CHEL lines of Chinese hamster cells and HeLa cells. Sodium butyrate enhances the chromatin sensitivity to nucleases and removes the radioprotective effect of cysteamine as measured by the yield of cells with chromosome aberrations. As is indicated by changes in the intensity of fluorescence of the DNA-ethidium bromide complex, measured by laser flow cytometry, the protective agent decreases the binding of the dye with both irradiated and nonirradiated DNA whereas ionizing radiation and sodium butyrate increase thereof. It is concluded that the radioprotective effect of cysteamine depends in its ability to reduce the susceptibility of DNA to nucleases.     

Different changes of plasma membrane beta-adrenoceptors in rat heart after chronic administration of propranolol, atenolol and bevantolol

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.     

Influence of adrenoceptors on thrombin-induced phosphoinositide metabolism in rat platelets

Stimulation of washed rat platelets with thrombin resulted in an increased turnover of phosphoinositides. Adrenaline and isoproterenol both inhibited thrombin-induced phosphatidic acid formation in a dose-dependent manner. Inhibitory responses of both compounds were blocked by a beta-adrenoceptor antagonist. However, isoproterenol was a more potent inhibitor than adrenaline. Addition of a selective alpha2-adrenoceptor antagonist potentiated the inhibitory effect of adrenaline up to the level observed with isoproterenol. Prestimulation of beta-adrenoceptors with isoproterenol, followed by addition of adrenaline (or noradrenaline) markedly diminished the inhibitory effect induced by the full beta-adrenoceptor agonist. Our results indicate that, in rat platelets, catecholamines are able to counteract, via alpha2-receptors, the beta-adrenoceptor-mediated inhibition of thrombin-induced phosphatidic acid formation. This suggests that catecholamines, by controlling cAMP level, may modulate phospholipase C activity and thereby platelet reactivity.     

Characterization of beta-adrenoceptors responsible for venom production in the venom gland of the snake Bothrops jararaca

We have shown that the stimulation of beta-adrenoceptors is an important step in venom production in the Bothrops jararaca venom gland. In the present study, the pharmacological profile of the beta-adrenoceptor present in Bothrops jararaca venom gland was characterized by radioligand binding assay and by the ability of isoprenaline to promote accumulation of cyclic AMP in dispersed secretory cells. In both cases, the venom glands were obtained from non-extracted snakes (quiescent stage) or from snakes which venom was extracted 4 days before sacrifice (venom production stimulated stage). [125I]-iodocyanopindolol ([125I]-ICYP) bound to extracted gland membranes in a concentration-dependent and saturable manner, but with low affinity. Propranolol, beta1- or beta2-selective adrenoceptors ligands displaced the [125I]-ICYP binding with low affinity, while selective beta3-adrenoceptor ligands did not displace the [125I]-ICYP binding. The displacement of [125I]-ICYP by propranolol was similar in non-extracted and extracted glands, showing the presence of beta-adrenoceptors in both stages. In dispersed secretory cells of non-extracted glands, isoprenaline (1 microM) increased the cyclic AMP production and propranolol (10 microM) was able to block this effect. On the other hand, in extracted glands, isoprenaline had no effect. The results suggest that the beta-adrenoceptors present in the Bothrops jararaca venom glands are different from those (beta1, beta2 or beta3) described in mammals, but are coupled to the Gs protein, like the known beta-adrenoceptor subtypes. Moreover, previous in vivo stimulation of venom production desensitizes the beta-adrenoceptors system and, although the receptors could be detected by binding studies, they are not coupled to the Gs protein, indicating that beta-adrenoceptors stimulation contributes to the initial steps of venom synthesis.     

The mechanism of the radioprotective action of sulfolane]

As a result of radiolysis of sulfolane, that has a radioprotective action, sulfonyl anion-radicals and hydrosulfonyl radicals are produced, subsequent transformations of which lead to complete regeneration of sulfolane. On the other hand, some other five-membered cyclic sulfones, that have no radioprotective action, are irreversibly depleted in reactions with water radiolysis products. Sulfolane is suggested to protect from the effect of e(-)aq and H. and to be ineffective in protecting against OH. radicals.     

Gi affects the agonist-binding properties of beta-adrenoceptors in the presence of Gs

Pertussis-toxin-catalyzed ADP-ribosylation of Gi in S49 membranes, but not in S49AC- membranes, which lack Gs, induces a threefold reduction of isoproterenol affinity to the beta-adrenoceptors. A similar treatment of turkey erythrocyte membranes, which are devoid of functional Gi, has no effect on beta-agonist affinity to their beta-adrenoceptors. Non-hydrolyzable analogs such as GTP[S] induce a larger decrease in beta-adrenoceptor affinity in S49 cells towards the agonist isoproterenol as compared to pertussis-toxin-catalyzed ADP-ribosylation of Gi. These results suggest that Gi affects beta-adrenoceptor affinity to its agonist and that this interaction requires the presence of Gs. It seems, therefore, that Gi physically interacts with Gs to exert its effects on the receptor and probably on adenylate cyclase as well. Our ability to detect (a) the effect of pertussis-toxin-catalyzed ADP-ribosylation in S49 cells on beta-agonist affinity and (b) the quantitative difference between the effect of pertussis toxin (approx. threefold) and GTP[S] (fivefold to sevenfold) depends on the use of a simple but rigorous method to study in detail the affinity of beta-agonists to their receptors. This method seems to be superior to the analysis of displacement curves as a means to examine receptor-ligand interactions.     

Effect of electron and steric interactions on the radioprotective properties of indolylalkylamines

Molecular mechanisms of radioprotective action of some substituted indolylalkylamines are discussed in terms of the statistical correlation analysis. It was established that electron and steric properties of the substituents are the factors influencing the radioprotective efficiency of these compounds. It was shown that the correlation obtained, relating the structure of the compounds to the radioprotective effect, may be applied in studies of the mechanism of action of the preparations and for the purposeful synthesis of new compounds.     

Effects of salbutamol and butoxamine on the human fat cell adenylate cyclase

In order to characterize the beta-adrenoceptors coupled to the human fat cell adenylate cyclase more extensively the effects of the beta 2-selective agonist salbutamol on basal and isoproterenol-stimulated rates of cAMP-accumulation were studied. Although exhibiting only low intrinsic activity salbutamol displayed only slightly lower affinity towards the beta-adrenoceptors linked to the human fat cell adenylate cyclase than isoproterenol. In addition, the beta 2-selective antagonist butoxamine was slightly more potent in inhibiting the isoproterenol-stimulated fat cell enzyme than the cardioselective beta-blocking agent practolol. These results further emphasize the difficulties in classifying the lipolytic response of adipose tissue to beta-adrenergic agonists and antagonists within a uniform beta-receptor theory.     

Radioprotective action of catecholamines on a Chinese hamster fibroblast culture

The radioprotective ability of adrenaline, noradrenaline and isoproterenol in various concentrations has been studied in experiments on cultured Chinese hamster fibroblasts in vitro. Radioprotective effect of isoproterenol is pronounced at 10(-8)M concentration; adrenaline and especially noradrenaline are effective at higher concentrations. The antagonist of beta-adrenergic receptors propranolol blocks the radioprotective effect of catecholamines on cells. The beta-adrenergic mechanism of catecholamines radioprotective action on the mammals cells is under discussion.     

Effects of intraluminal administration of adrenoceptor agonists on transisthmic flow in the rabbit oviduct

By perfusing the isthmic segment of the rabbit oviduct in vivo at low hydrostatic pressures with Ringer's solution (+38 degrees C), the resistance of the isthmic segment could be measured quantitatively using a drop-recording system. Transisthmic flow, from mid-tube to the uterine end, was thus studied in 45 animals, 48 h after human chorionic gonadotropin (hCG) injection. Stimulation of alpha-adrenoceptors by administration of norepinephrine or phenylephrine to the perfusate resulted in an immediate reduction or cessation of the basal flow. The duration of this effect varied dose-dependently. The administration of beta-adrenoceptor agonists (isoprenaline, prenalterol or terbutaline) markedly increased transisthmic flow. The beta 2-adrenoceptor agonist terbutaline was considerably more potent than the other beta-agonists. Thus, it seems that the isthmic contraction caused by adrenergic luminal stimulation is mediated via alpha-adrenoceptors, while the isthmic dilation is mediated via beta-adrenoceptors, mainly of the beta 2-type.     

Coexistence of at least three distinct beta-adrenoceptors in human internal mammary artery.

The internal mammary artery (IMA) is currently the preferred conduit for myocardial revascularization. However, perioperative vasospasm and a hypoperfusion state during maximal exercise may limit its use as a bypass graft. The mechanism of spasm has not been clearly defined. Since beta-adrenoceptor activation plays a major role in vasorelaxation, the present study was carried out to investigate the beta-adrenoceptor responsiveness of human IMA smooth muscle. Isoproterenol produced a concentration-dependent relaxation in endothelium-denuded IMA segments, precontracted with phenylephrine (maximal relaxation 46.33+/-5.45%). Atenolol (10(-6)M) and propranolol (2x10(-7)M) inhibited isoproterenol-induced relaxation. While atenolol produced partial inhibition, propranolol caused a complete inhibition in a majority of the segments and a partial inhibition in a minority. BRL 37344, a selective beta 3-adrenoceptor agonist, produced a concentration-dependent relaxation in phenylephrine-precontracted rings of endothelium-denuded IMA (maximal relaxation 40.35+/-4.07%). Cyanopindolol, a beta-adrenoceptor partial agonist, produced a marked relaxation (58.65+/-6.2%) in endothelium-denuded IMA rings, precontracted with phenylephrine. Cyanopindolol-induced relaxation was resistant to blockade by propranolol (2x10(-7)M). Spontaneous contractions of IMA rings were also observed in some cases that were inhibited by isoproterenol and BRL 37344. This observation implies the important role of beta-adrenoceptor activation in prevention of human IMA spasm. The results obtained in present study indicate that human IMA smooth muscle possesses an atypical beta-adrenoceptor together with beta1- and beta2-adrenoceptors. Regarding the relaxation induced in IMA rings by adding BRL 37344, the possible identical entities of IMA atypical beta-adrenoceptors and beta 3-adrenoceptors are suggested.