Claudia Perez Leiros

Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor-inotropic response

1 In this paper we have determined the different signalling pathways involved in muscarinic acetylcholine receptor (AChR)‐dependent inhibition of contractility in rat isolated atria. 2 Carbachol stimulation of M2 muscarinic AChRs exerts a negative inotropic response, activation of phosphoinositide turnover, stimulation of nitric oxide synthase and increased production of cyclic GMP. 3 Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase, shifted the dose‐response curve of carbachol on contractility to the right. These inhibitors also attenuated the muscarinic receptor‐dependent increase in cyclic GMP and activation of nitric oxide synthase. In addition, sodium nitroprusside, isosorbide, or 8‐bromo cyclic GMP, induced a negative inotropic effect, increased cyclic GMP and activated nitric oxide synthase. 4 These results suggest that carbachol activation of M2 AChRs, exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This in turn, triggers cascade reactions involving calcium/calmodulin and protein kinase C, leading to activation of nitric oxide synthase and soluble guanylate cyclase.

Cholinergic response of isolated rat atria to recombinant rat interferon-γ

Addition of recombinant rat interferon-gamma (IFN-gamma) to beating rat atria decreased the contractile strength in a dose-dependent manner. The effect was specific of IFN-gamma since it was abrogated by monoclonal anti-rat IFN-gamma. It required the activation of the cholinergic system of the heart as inhibition of both nicotinic (10(-7) M hexametonium) and muscarinic cholinoceptors (10(-7) M atropine) prevented the reaction. Hemicholinium (2 x 10(-5) M) and tetrodotoxin (5 x 10(-7) M) also reduced the response. Likewise, IFN-gamma potentiated the action of the muscarinic agonist carbachol. IFN-gamma simulated the biological effect of cholinergic agonists because: (a) it increased cGMP formation; (b) it decreased cAMP formation; and (c) it reduced heart contractility at doses that can be considered physiologic. IFN-gamma also modified the muscarinic receptor by interfering with the binding of the radiolabelled antagonist quinuclidinyl benzilate [( 3H]QNB). It is suggested that IFN-gamma binding to IFN-gamma receptors in the heart may lead to a cholinergic response by interaction of both receptor systems on the surface of atrial cells.

β-Adrenergic Cardiac Antibody in Autoimmune Myocarditis

Balb/c mice were immunized with homologous heart in complete Freunds adjuvant to induce autoimmune myocarditis. The myocarditis was characterized by lymphomononuclear infiltration, electrocardiographic abnormalities and antimuscle antibodies by indirect immunofluorescence. In this paper, we demonstrate that the IgG present in autoimmune myocarditis mice is able to bind to beta-adrenoreceptors of the heart and also induce a biological effect inhibiting the contractile action of exogenous norepinephrine. Auto-immune IgG inhibited the binding of (3H)-dyhidroalprenolol to a beta-adrenergic receptor of purified myocardial membranes behaving as non-competitive inhibitor. This IgG also exerted a non-competitive inhibition upon the mechanical effect of exogenous norepinephrine. The recognition appears to be organ specific, because the autoimmune myocarditis IgG did not bind to beta-lymphocyte, lung and fat adrenoreceptors. The autoimmune IgG inhibited the stimulatory action of isoproterenol on cAMP levels, behaving as a beta-adrenergic antagonist.

Characterization of (-)-(3H)-DHA binding to intact mouse lymphocytes: Effect of experimental autoimmune orchitis on β-adrenoceptor expression

Autoimmune orchitis induced an increment in the beta-adrenoceptor populations in intact mouse lymphocytes, depending on their source. Characterization of (-)-(3H)-DHA specific binding to intact normal cells indicate that thymic cells do not have the ability of binding a beta-adrenergic ligand. In contrast, spleen and lymph node cells showed a homogeneous population of beta-adrenergic receptors. (-)-(3H)-DHA binding was a rapid, reversible and stereospecific process. Competition experiments indicated the order of potency of the agonists and led to their definition as being of the beta 2-adrenoceptor subtype. Saturation assays and Scatchard analysis indicated a single class of binding sites (being free of allosteric or cooperative interaction) in both control and immune cells. However, spleen and lymph node cells from mice hyperimmunized with testicular preparations showed a marked increase in the number of beta-adrenoceptor sites with no changes in chi d. These results emphasize the use of lymphocytes possessing a homogeneous population of beta 2-adrenoceptors as an easily available model for monitoring beta-adrenoceptors in disease.

Effect of histamine in autoimmune myocarditis mice

The contractile effect of histamine, as well as the H1 receptor population and H2 receptor-mediated cAMP production, were measured in cardiac tissue from control normal and autoimmune myocarditis mice. Histamine triggered positive chronotropy and negative inotropy at high concentrations in both control and autoimmune auricles, H2 receptors being the most important mediator of these responses. In contrast, in atria from autoimmune myocarditis mice, histamine at lower concentrations caused positive inotropy and negative chronotropy. These effects, not verified in the normal control atria, are mediated by H1 receptors. The expression of H2 and H1 receptors mediating the cardiac response to histamine was evaluated through histamine-stimulated cAMP level and binding of [3H] mephyramine, respectively. Both control and autoimmune myocardium were able to increase cAMP levels, an effect that was inhibited by H2 antagonist drug. The amount of cAMP was significantly higher in control myocardium than in those from autoimmune ones. Saturable binding of [3H] mephyramine occurs in autoimmune myocardium, with distinct high and low affinity binding sites. In control myocardium non-saturable binding was detected. These results suggest that H1 and H2 receptors coexist in heart from autoimmune myocarditis mice, whereas only H2 receptors are present in myocardium from control mice. The presence of H1 receptors in autoimmune myocardium could be an important factor in the regulation of its physiological behaviour.

Expression of α-adrenoceptors in a human transformed lymphoblastoid cell line

Abstract The presence of α-adrenergic receptors (absent in normal lymphocytes) has been demonstrated in transformed human lymphocytes of the Raji cell line. Binding properties of β-adrenergic receptors were similar to those reported for normal lymphocytes. A single population of α 2 -adrenergic receptors was characterized in intact Raji lymphoblasts by binding and saturation assays with the α 2 -adrenergic antagonist yohimbine. Competition curves with [ 3 H]yohimbine indicate the presence of typical α 2 -adrenoceptors. Reaction of Raji with the α 2 -adrenergic agonist clonidine (10 −6 M) stimulated their growth rate. In contrast, the α 1 -adrenergic agonist methoxamine (10 −6 M) had no effect. Previous work indicates that Raji can actively produce thromboxanes (TX) and that these decreased atrium contractility. In agreement with these results and with the binding studies, it is now shown that clonidine stimulation enhanced the negative inotropic effects of Raji on isolated rat atria. This reaction was prevented by incubation of Raji with yohimbine (10 −6 M) but not with the α 1 -adrenergic antagonist prazosin (10 −6 M) or the β-adrenergic antagonist propranolol (10 −7 M). The biologic effect of Raji on rat atria was probably due to production of cyclooxygenase metabolites of arachidonic acid, because it was blocked by preincubation of the cells with the cyclooxygenase inhibitors indomethacin (10 −6 M) and aspirin (10 −4 M) or the thromboxane synthetase inhibitors nictindol (10 −5 M) and imidazole (10 −4 M). The presence of α-adrenoceptors on transformed Raji cells may play a role in the regulation of cell growth and biologic activity, since α 2 -adrenergic stimulation can change the balance of the intracellular signals involved in triggering cell division and function.

Increases in cyclic AMP levels couple to H1 receptors in atria from autoimmune myocarditis mice

We have previously shown that myocardium from experimental autoimmune myocarditis expresses H1 receptors not present in normal mice heart. ThEA acting via H1 receptors, augments cyclic AMP production in atria from autoimmune myocarditis mice without any effect on atria from control mice. Addition of mepyramine before ThEA caused cyclic AMP levels to fall to a level similar to basal, confirming the H1 receptor participation. Histamine at low concentrations mimicked the ThEA action on H1 receptor-stimulation of cyclic AMP production by autoimmune myocardium. The fact that the inhibition of phospholipase C blocked the cyclic AMP stimulation by ThEA, supports the assumption that this action is secondary to receptor-mediated hydrolysis of phosphoinositides, generating some oxidative metabolites (IP3-DAG), which in turn may be responsible for the cyclic AMP effect. So, the inhibition of protein kinase C and calcium/calmodulin partially prevented the stimulatory action of ThEA on cyclic AMP levels in autoimmune myocardium, suggesting that both pathways are implicated in this effect. Data shows that the stimulation of H1 receptors by specific agonist in atria from autoimmune myocarditis mice, augments the cyclic AMP, requiring the hydrolysis of phosphoinositide cycle. The role of this cyclic AMP augmentation in myocardium from autoimmune myocarditis mice, will provide a basis to assess the role of this second messenger as an important factor in the regulation and/or modulation of the physiological behaviour of the heart in the course of autoimmune myocarditis.