Contribution of 4-hydroxy-2,3-trans-nonenal to the reduction of beta-adrenoceptor function in the heart by oxidative stress

Oxidative stress reduces adenylate cyclase activity and also the maximal response to beta-adrenoceptor stimulation in the rat heart, while beta-adrenoceptor density is not affected or increased. Since free sulfhydryl groups are essential to beta-adrenoceptor function and the sulfhydryl reactive substance 4-hydroxy-2,3-trans-nonenal (HNE) is responsible for part of the effects of oxidative stress, the effect of HNE on beta-adrenoceptor function in field stimulated left atria of the rat was determined. To this end field stimulated atria were incubated with 10 microM, 100 microM and 1 mM HNE for 25 min. After removing the excess of HNE, beta-adrenoceptor function was determined by measuring the positive inotropic response to (-)-isoproterenol. It was found that 10 microM HNE had no effect on beta-adrenoceptor function, whereas 100 microM HNE reduced the maximal effect to (-)-isoproterenol without affecting the pD2 (-log EC50). At these concentrations, HNE had no effect on either beta-adrenoceptor density or on c-AMP production. After 1 mM HNE, the atria stopped contracting. Since the effects of the synthetic thiol inactivator N-ethyl maleimide were similar to those of HNE, it was concluded that the reduction of beta-adrenoceptor function by HNE is probably the result of alkylation of free sulfhydryl groups. Our results indicate that the reduction of adenylate cyclase activity by oxidative stress is not mediated by the production of HNE, however oxidative stress and HNE both reduce the maximal response to beta-adrenoceptor stimulation.     

Differences and Similarities in Muscarinic Receptors of Rat Heart and Retina: Effects of Agonists, Guanine Nucleotides, and N-Ethylmaleimide

Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N-ethylmaleimide decreases agonist affinity for the high-affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of high- and low-affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low-affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high-affinity binding sites in retina. Third, the "partial agonist" pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high- and low-affinity sites and to effect a biological response.     

Adrenergic-cholinergic interactions in left atria: interaction of carbachol with alpha- and beta-adrenoceptor agonists

The purpose of the present investigation was to determine the nature of the functional interaction of muscarinic agonists with cAMP-generating and cAMP-independent agonists in left atria. Negative inotropic responses of rabbit isolated left atrial strips to the muscarinic agonist carbachol were measured in the absence and presence of equi-active inotropic doses of the beta-adrenoceptor stimulant isoproterenol (Iso), the mixed alpha- and beta-adrenoceptor stimulant phenylephrine (PE) plus 1 microM timolol to block the beta-receptor mediated component of its response, and elevated extracellular Ca2+. Carbachol produced dose-dependent negative inotropic responses in left atrial strips, which were much greater than control in the presence of either Iso, or PE plus timolol. However, carbachol responses were of a similar magnitude to the control in the presence of elevated extracellular Ca2+. In the presence of timolol, PE had no significant effect on cAMP levels in left atrial strips, and inotropic responses to carbachol alone and in combination with PE plus timolol were accompanied by significant increases in cGMP levels but no change in cAMP levels. Carbachol attenuated Iso-induced increases in cAMP levels, but decreases in left atrial tension were proportionally greater than the decreases in cAMP levels produced by carbachol in the presence of Iso. These results suggest that the antiadrenergic effects of muscarinic receptor stimulation may occur by a different mechanism in left atria than has been previously reported in ventricular muscle. While the nature of this mechanism is unknown, it may involve antagonism by muscarinic agents of both alpha- and beta-adrenoceptor mediated increases in Ca2+ influx.     

Responses mediated via beta 1, but not beta 2-adrenoceptors, exhibit hypothermia-induced supersensitivity

The beta-adrenoceptor-mediated responses of various isolated tissues from the guinea-pig were examined at bath temperatures of 38 and 30 degrees C. The positive inotropic responses to orciprenaline of paced left atria and papillary muscles were potentiated at the lower bath temperature, as indicated by a significant shift of the dose-response curve to the left. A similar hypothermia-induced supersensitivity was observed for the positive chronotropic response of right atria. The beta-adrenoceptor-mediated inhibition of coaxially stimulated ileum in response to orciprenaline also exhibited supersensitivity at 30 degrees C. In contrast, the relaxation of lung strips was not altered at the lower bath temperature. The relaxant responses of potassium-contracted uterine strips from untreated guinea-pigs or progesterone-dominated guinea-pigs and rats also failed to reveal any hypothermia-induced supersensitivity. The responses of lung and uterine strips are mediated via beta-adrenoceptors of the beta 2-type, whereas they are of the beta 1-type in the cardiac preparations and ileum. Therefore, these results suggest that hypothermia-induced supersensitivity occurs only at the beta 1-adrenoceptors, indicating a fundamental temperature-dependent difference between the two receptor types.     

Effect of glucose on potassium contracture tension in rat heart

The effect of substrates on potassium contracture tension of the isolated rat ventricle strip was investigated. The contracture tension magnitude of ventricle strips exposed to potassium rich medium was markedly greater with medium containing pyruvate or acetate than with glucose as the substrate. The effect of substrates on contracture tension was not related to their ability to maintain the ATP stores of the heart, for there was not a significant difference in the ATP levels in ventricle strips incubated in medium containing pyruvate, pyruvate + glucose, or glucose. Glucose reduced the K-contracture tension magnitude of heart strips suspended in medium containing pyruvate or acetate as substrate; 3-0-methylglucose and 2-deoxy-D-glucose did not have this action on K-contracture tension. The reduction of K-contracture tension by glucose was inhibited by iodoacetate and fluoride. Under anaerobic conditions, 50 mM glucose significantly reduced the K-contracture tension of ventricle strips suspended in pyruvate medium. The findings of this study suggest that glucose metabolism has an action on K-contracture tension in cardiac muscle that is not shared by acetate or pyruvate.     

Characterization of myometrial desensitization to beta-adrenergic agonists

Continuous exposure of ovine myometrial strips exposed to isoproterenol (10 microM) resulted in only transient inhibition with contractions returning within 60 min. Rechallenging these strips with isoproterenol failed to induce inhibition, confirming the occurrence of desensitization. In contrast, exposure of myometrial tissue to isoproterenol for only 5 min did not result in desensitization. Myometrial strips exposed to isoproterenol demonstrated a significant increase in cAMP content associated with inhibition of contractile activity and a subsequent fall in cAMP content upon desensitization. Elevation of endogenous cAMP levels by either inhibition of cAMP-dependent phosphodiesterase activity (0.5 mM isobutylmethylxanthine, in ovine strips) or direct activation of adenylyl cyclase (10 microM forskolin, in rat strips) induced a rapid and significant inhibition of myometrial contractile activity in desensitized tissue. Scatchard analysis of the binding of the beta-adrenoceptor antagonist, [125I]iodocyanopindolol, revealed a significant reduction in the concentration of beta-adrenergic receptors (but no change in binding affinity) in desensitized myometrial tissue. Incubation of desensitized tissue with fresh buffer for 3 h induced only a partial recovery in responsiveness to isoproterenol. These data suggest that prolonged, but not acute, exposure of the myometrium to beta-adrenergic agonists induces a state of desensitization that is associated with a down-regulation of beta-adrenoceptors but maintenance of postreceptor function.     

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.     

Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-L-arginine and N-iminoethyl-L-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.     

Alpha-tocopherol pretreatment improves endothelium-dependent vasodilation in aortic strips of young and aging rats exposed to oxidative stress

Acetylcholine-induced, endothelium-dependent relaxation of norepinephrine-precontracted aortic strips, was severely impaired after exposure to a hypoxanthine/xanthine oxidase reaction generating oxygen radicals. This effect was more evident in aortic strips of aging rats (24 months old) in comparison to young rats (3 months old). The addition of authentic ·NO (1 M) completely relaxed aortic strips exposed to oxidative stress both in young and aging rats. In vitro EPR measurements showed that the ·NO signal was reduced by enzymatic O₂-generating reaction.The activity of a partial purified preparation of constitutive NO synthase from rat cerebellum was significantly decreased after exposure to exogenous oxygen radicals. Pretreatment of aortic strips with 100 M alpha-tocopherol-phosphate, produced a significant improvement of acetylcholine-dependent relaxation in the aortic strips exposed to oxidative stress, particularly in the aged vessel. The content of malondialdehyde in aortic tissue did not change after oxidative stress or alpha-tocopherol pretreatment. Alpha-tocopherol was unable to recover the NO synthase activity depressed in vitro by hypoxanthine/xanthine oxidase reaction. This study confirms that an oxidative stress impairs the endothelium-mediated vasodilation. Alpha-tocopherol pretreatment protects the vessel against this damage. The mechanism of action of alpha-tocopherol is unknown, but seems unrelated to an antioxidant activity.Abbreviations ACh acethylcholine - EPR electron paramagnetic resonance - ROS reactive oxygen species - MDA malondialdehyde - NE norepinephrine - cNOS constitutive nitric oxide synthase     

Beta 2-adrenoceptor response in the rat uterus at the end of gestation and after induction of labor with RU 486

To study the relationship between the progesterone environment and beta-adrenoceptors in the myometrium, rats were treated with the antiprogestin RU 486 (10 mg per rat) at 08:30 h on day 21 of gestation. Under these conditions, more than 60% of animals delivered within 24 h after this treatment, while none of the control animals delivered within the same time period. beta-Adrenoceptors were identified using the radiolabeled antagonist (-)-[125I] iodocyanopindolol. The density (Bmax approximately 33-45 fmol/mg protein) and the affinity (KD approximately 0.105-0.106 nM) were not changed (during the late stages of gestation) in RU 486 treated rats compared with control rats. These results were correlated with the relaxation of longitudinal and circular strips of myometrium placed in high KC1 medium and exposed to beta-adrenoceptor agonists. The adrenoceptors implicated in the relaxation of myometrial strips were mainly of the beta 2-subtype. There was no difference in their affinity between control and RU 486 treated rats. Mean pA2 values were 8.46 for propranolol and 8.27 for ICI 118-551 against salbutamol. Altogether these results indicate in the rat that the blockade of the action of progesterone at its receptor site by RU 486 did not modify either the affinity or the sensitivity of beta 2-adrenoceptors in the myometrium, although it induced parturition.     

Pharmacological evaluation of plasma membrane beta-adrenoceptors in rat hearts using the tissue segment binding method

This study evaluates beta-adrenoceptors in rat atria and ventricle using the tissue segment binding method and compares the results with those obtained using conventional homogenate binding assays. In studies with tissue segment binding, the hydrophilic radioligand [(3)H]-CGP12177 selectively bound to plasma membrane beta-adrenoceptors, and the B(max) levels were significantly higher than those obtained with homogenate binding. However, both binding approaches revealed similar proportions of beta(1)- and beta(2)-adrenoceptors. The regional distribution of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts were also determined using tissue segment binding. Abundance of beta-adrenoceptors and proportion of beta(1)-adrenoceptors were higher in atria than in ventricle, but there was no significant difference between right and left atria or within ventricle (right and left ventricle free walls, apex, and interventricular septum). To establish the ability of the tissue segment binding method to study beta-adrenoceptor regulation such as the internalization of receptors, the effect of prolonged exposure of rat ventricle to (-)-isoprenaline was also investigated by using tissue segments and homogenate binding. Incubation with (-)-isoprenaline for 1 h in vitro caused a concentration-dependent decrease in the density of beta-adrenoceptors, predominantly beta(2)-adrenoceptors, when assessed with tissue segment binding method. In contrast, the subtype-specific change after treatment with (-)-isoprenaline was not detected using homogenate binding. In summary, the tissue segment binding method with [(3)H]-CGP12177 enables a more precise quantitation of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts and is suitable for studying their regulation.     

Membrane Microviscosity Modulates μ-Opioid Receptor Conformational Transitions and Agonist Efficacy

The influence of membrane microviscosity on mu-opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH-SY5Y cells and from a C6 glioma cell line stably expressing the rat mu-opioid receptor (C6mu). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) for the mu-opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis-vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol-receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS-treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist-stimulated GTPase activity in control and CHS-modified membranes. These findings support a regulatory role for membrane microviscosity in receptor-mediated G protein activation.     

Glutamate induces formation of free radicals in rat brain synaptosomes

The influence of glutamate and agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes was investigated using the fluorescent dye DCFDA. Glutamate at concentrations of 100 μM and 1 mM increased the production of reactive oxygen species. This phenomenon was eliminated by removing calcium from the incubation medium. Addition of NMDA (100 μM) or kainate (100 μM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.     

Relationships Between Ligand Affinities for the Cerebellar Cannabinoid Receptor CB1 and the Induction of GDP/GTP Exchange

The hypothesis of these studies is that ligand efficacy at the neuronal CB1 receptor is dependent on the ratio of ligand affinities for the active and inactive states of the receptor. Agonist efficacy was determined in rat cerebellar membranes using agonist-induced guanosine 5'-O-(3-[35S]thiotriphosphate) binding; efficacy was variable among the CB1 agonists examined. Ligand affinities for the active and inactive state of the CB1 receptor were determined by competition with [3H]CP55940 and [3H]SR141716A in the presence of 5'-guanylylimidodiphosphate, respectively. All of the agonists investigated had a higher affinity for the active state than the inactive state. The fraction of CB1 receptors in the active state at a maximally effective concentration was calculated for each agonist and was found to correlate significantly with agonist efficacy. These studies demonstrate that the CB1 receptor of the cerebellum can assume an active conformation in the absence of agonist and that the variability in efficacy among CB1 receptor agonists can be explained by the relative affinities of these ligands for the CB1 receptor in the active and inactive states.     

Influence of endogenous catecholamines on responses of rat lung to beta-adrenergic agonists

Relaxation responses to the beta-adrenoceptor agonists: isoprenaline (non selective), salbutamol (beta 2-selective) and noradrenaline (plus phentolamine 10(-5) M) (beta 1-selective) have been obtained on rat lung parenchymal strips in the absence and presence of pargyline and tropolone (monoamino-oxidase and catechol-O-methyltransferase inhibitors), cocaine (neuronal uptake blocking agent), corticosterone (extraneuronal uptake inhibitor) as well as in reserpinized rat. Responses to these beta-adrenergic agonists were not potentiated in the presence of any of these inhibitors. This indicates that endogenous catecholamines, enzymatic or uptake processes, do not modulate beta-adrenoceptor mediated responses of rat lung strip and demonstrates that there is no correlation between neuronal uptake/beta 1-adrenoceptors and extraneuronal uptake/beta 2-adrenoceptor mediated responses, as had previously been suggested.     

Effects of [Ca2+]o on contractility in the anoxic cardiac muscle of mammal and fish

Electrically paced atrial strips of hearts from rat and rainbow trout were exposed to increasing extracellular Ca2+ concentration, [Ca2+]o. This resulted in increases in the peak force in oxygenated atria from both species. During anoxia this response was suppressed for the rat, but accentuated for trout atrium.     

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content

The cardiac neuronal norepinephrine (NE) transporter (NET) in sympathetic neurons is responsible for uptake of released NE from the neuroeffector junction. The purpose of this study was to assess the chamber distribution of cardiac NET protein measured using [(3)H]nisoxetine binding in rat heart membranes and to correlate NE content to NET amount. In whole mounts of atria, NET was colocalized in nerve fibers with tyrosine hydroxylase (TH) immunoreactivity. NE content expressed as micrograms NE per gram tissue was lowest in the ventricles; however, NET binding was significantly higher in the left ventricle than the right ventricle and atria (P < 0.05), resulting in a significant negative correlation (r(2) = 0.922; P < 0.05) of NET to NE content. The neurotoxin 6-hydroxydopamine, an NET substrate, reduced NE content more in the ventricles than the atria, demonstrating functional significance of high ventricular NET binding. In summary, there is a ventricular predominance of NET binding that corresponds to a high NE reuptake capacity in the ventricles, yet negatively correlates to tissue NE content.     

Altered inotropic reactivity in diabetic rabbit right ventricular myocardium

Alloxan monohydrate was used to induce diabetes in rabbits, which were maintained for a 3-month period with or without daily insulin replacement along with age-matched controls. Isolated right ventricular myocardial strips were used to generate dose-response curves to isoproterenol, forskolin, and Bay K 8644. Basal developed force was significantly elevated in diabetic ventricular strips. While isoproterenol acted as a full inotropic agonist, diabetic preparations revealed a consistent but insignificant decrease in the maximum developed force. While both sensitivity to isoproterenol and beta-adrenoceptor density were decreased in preparations from diabetic rabbits, there was no associated increase in circulating plasma catecholamines. In contrast, forskolin and Bay K 8644 were partial agonists in control preparations but full inotropic agonists in diabetic preparations, demonstrating significant increases in maximum developed force. This hyperresponsiveness was not associated with altered calcium channel density. Finally, insulin replacement reduced or prevented all diabetic-related changes. These data indicate that the hyperresponsiveness to forskolin and Bay K 8644 represents an altered utilization of intracellular calcium in the diabetic rabbit, converting them into full agonists similar to isoproterenol. The decrease in sensitivity to isoproterenol correlated with a decrease in beta-adrenoceptor density but not elevated circulating catecholamines as previously observed in diabetic rats.