The beta-adrenergic receptor in human lymphocytes: subclassification by the use of a new radio-ligand, (+/-)-125 Iodocyanopindolol

(±)?¹²⁵Iodocyanopindolol (ICYP), a new radio-ligand with high affinity and specificity to β-adrenoceptors was used to identify and characterized β-adrenergic receptors in human lymphocytes. Binding of ICYP was saturable with 1.56 ± 0.2 fmol ICYP specifically bound/10⁶ cells at maximal occupancy of the sites and of high affinity (K_D=57 ± 7.1pM, N=4. In contrast to ¹²⁵Iodohydroxybenzylpindolol ICYP-binding was not affected by phentolamine (up to 10^?4M) or serotin (up to 10^?5M). Analysis of inhibition of ICYP-binding via a pseudo-Scatchard-plot (“Hofstee-plot”) by β_1-selective (practocol, metaprolol) and β_2-selective (IPS 339, zinterol) adrenergic drugs resulted in linear plots suggesting the existence of a homogeneous population of β-adrenergic receptorsin human lymphocytes. From the resulting K_D-values for practolol (16.8 μM), metoprolol (4.11 μM), zinterol (0.08 μM) and IPS 339 (0.002 μM) is concluded that the β-adrenergic receptor present in human lymphocytes is of the β₂-subtype. According to its low non-specific binding and its high specificity to β-adrenergic receptors ICYP appears to be an ideal ligand for long-term studies on the regulation of β-adrenergic receptors of human lymphocytes.     

INFLUENCE OF COLD EXPOSURE ON THE ACTIVITIES OF SOLUBLE AND MEMBRANE-BOUND DOPAMINE β-HYDROXYLASE IN RAT HEART VESICLES

Abstract— A fraction containing noradrenaline storage vesicles of the sympathetic nerve terminals in the rat heart was obtained by differential centrifugation. In this preparation, 17% of the dopamine β-hydroxylase was present in a soluble form. Cold exposure (3°C) for periods from 5 to 30 min led to an increase in the activity of soluble dopamine β-hydroxylase by about 50%, while the activity of membrane-bound dopamine β-hydroxylase was simultaneously decreased by approx 30%. The nor-adrenaline content of the vesicles rose concomitantly with the increase in the activity of soluble dopamine β-hydroxylase. This rise in noradrenaline content was caused by an enhanced synthesis and not by an alteration in the subcellular distribution. The results are discussed with respect to the fate of dopamine β-hydroxylase during enhanced sympathetic nerve activity.     

Pressure overload and neurohumoral activation differentially affect the myocardial proteome

Treatment with monocrotaline causes pulmonary hypertension in rats. This results in severe pressure overload-induced hypertrophy of the right ventricles, whilst the normally loaded left ventricles do not hypertrophy. Both ventricles are affected by enhanced neuroendocrine stimulation in this model. We analyzed in this model load-induced and catecholamine-induced changes of right and left ventricular proteome by two-dimensional gel electrophoresis, tryptic in-gel digest, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All analyzed animals showed right ventricular hypertrophy without signs of heart failure. Changes of 27 proteins in the right and 21 proteins in the left ventricular myocardium were found. Given the hemodynamic features of this animal model, proteome changes restricted to the right ventricle are caused by pressure overload. We describe for the first time a potentially novel pathway (BRAP2/BRCA1) that is involved in myocardial hypertrophy. Furthermore, we demonstrate that increased afterload-induced hypertrophy leads to striking changes in the energy metabolism with down-regulation of pyruvate dehydrogenase (subunit beta E1), isocitrate dehydrogenase, succinyl coenzyme A ligase, NADH dehydrogenase, ubiquinol-cytochrome C reductase, and propionyl coenzyme A carboxylase. These changes go in parallel with alterations of the thin filament proteome (troponin T, tropomyosin), probably associated with Ca(2+) sensitization of the myofilaments. In contrast, neurohumoral stimulation of the left ventricle increases the abundance of proteins relevant for energy metabolism. This study represents the first in-depth analysis of global proteome alterations in a controlled animal model of pressure overload-induced myocardial hypertrophy.     

Impaired platelet activation in familial high density lipoprotein deficiency (Tangier disease)

ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, alpha2beta1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.     

Neutrophils Turn Plasma Proteins into Weapons against HIV-1

As a consequence of innate immune activation granulocytes and macrophages produce hypochlorite/hypochlorous acid (HOCl) via secretion of myeloperoxidase (MPO) to the outside of the cells, where HOCl immediately reacts with proteins. Most proteins that become altered by this system do not belong to the invading microorganism but to the host. While there is no doubt that the myeloperoxidase system is capable of directly inactivating HIV-1, we hypothesized that it may have an additional indirect mode of action. We show in this article that HOCl is able to chemically alter proteins and thus turn them into Idea-Ps (Idea-P = immune defence-altered protein), potent amyloid-like and SH-groups capturing antiviral weapons against HIV-1. HOCl-altered plasma proteins (Idea-PP) have the capacity to bind efficiently and with high affinity to the HIV-1 envelope protein gp120, and to its receptor CD4 as well as to the protein disulfide isomerase (PDI). Idea-PP was able to inhibit viral infection and replication in a cell culture system as shown by reduced number of infected cells and of syncytia, resulting in reduction of viral capsid protein p24 in the culture supernatant. The unmodified plasma protein fraction had no effect. HOCl-altered isolated proteins antithrombin III and human serum albumin, taken as representative examples of the whole pool of plasma proteins, were both able to exert the same activity of binding to gp120 and inhibition of viral proliferation. These data offer an opportunity to improve the understanding of the intricacies of host-pathogen interactions and allow the generation of the following hypothetical scheme: natural immune defense mechanisms generate by posttranslational modification of plasma proteins a potent virucidal weapon that immobilizes the virus as well as inhibits viral fusion and thus entry into the host cells. Furthermore simulation of this mechanism in vitro might provide an interesting new therapeutic approach against microorganisms.     

Beta2-adrenoceptor polymorphisms: relation between in vitro and in vivo phenotypes

Beta2-adrenoceptors are expressed in many cell types throughout the body and play a pivotal role in the regulation of the cardiac, pulmonary, vascular, endocrine and central nervous system. Recent studies have discovered that Beta2-adrenoceptor are polymorphic. Three common polymorphisms appear to influence receptor function: Arg16Gly, Gln27Glu, and Thr164Ile. In vitro studies of agonist-stimulation have shown that the Gly16 Beta2-adrenoceptors demonstrate enhanced agonist-promoted down-regulation, while Glu27 variants seem to be resistant. The Ile164 variant, on the other hand, demonstrates decreased responsiveness to agonist stimulation in vitro. However, the functional relevance and phenotypic consequence of such Beta2-adrenoceptor variants in vivo is still unclear. The aim of this review is therefore to provide an overview about the somewhat controversy in vitro, ex vivo and in vivo studies.     

Vascular dopamine receptors: Demonstration and characterization by in vitro studies

Substantial evidence has accumulated that in certain vascular beds dopamine produces its relaxant effect through stimulation of specific dopamine receptors. The goal of this review is to describe several in vitro models (perfused mesenteric vessels of the dog; renal, mesenteric, splenic, coronary and cerebral arterial strips of rabbits, dogs and cats; perfused kidney of the rat) recently developed to demonstrate such specific relaxations induced by dopamine and dopaminomimetics. On these models studies on structure-activity relationship for activation of the dopamine receptor resulted in the following order of potency for agonists: SK&F 38393 (partial agonist) greater than epinine greater than A-6, 7-DTN greater than or equal to dopamine greater than N, N-di-n-propyl-dopamine (partial agonist) greater than apomorphine (partial agonist). The dopamine receptor antagonists (+)-butaclamol, cis-alpha-flupenthixol, metoclopramide, droperidol and bulbocapnine were found to competitively antagonize dopamine induced relaxation. In addition, in two isolated organ systems (rabbit mesenteric artery, rat perfused kidney) stereospecificity of the vascular dopamine receptor was demonstrated with the isomers of butaclamol. With the development of several in vitro models demonstrating a specific antagonism against dopamine induced relaxation an important requirement for definition of a specific dopamine receptor if fulfilled according to classical pharmacological criteria. Thus, there can be do doubt on the existence of post-synaptic dopamine receptors mediating vasodilation in certain vascular tissues.     

Über die Synthese partiell benzylierter Zucker

1,2:5,6-Di-O-isopropylidene-α-D-allofuranose (1), 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2), and 1,2.3,4-di-O-isopropylidene-α-D-galactopyranose (3) have been separately treated in pyridine solution with trifluoromethanesulphonic anhydride, 2,2,2-trifluoroethanesulphonyl chloride, and pentaflucrobenzenesulphonyl chloride. Both 1 and 2 afforded the anticipated sulphonic esters. Although 3 also gave the 2,2,2-trifluoroethanesulphonic and pentafluorobenzenesulphonic esters, the reaction with trifluoromethanesulphonic anhydride yielded 6-deoxy-1,2:3,4-di-O isopropylidene-6-pyridino-α-D-galactopyranose trifluoromethanesulphonate.     

Specific antagonism by metoclopramide of dopamine-induced relaxation on isolated rabbit mesenteric arteries contracted with prostaglandin F2alpha.

On isolated rabbit mesenteric arteries pretreated with phenoxybenzamine (10-5M) and contracted with prostaglandin F_2α (PGF_2α) dopamine (10^?6M to 3×10^?4M) and isoprenaline (10-9M to 10-5M) caused a dose-related relaxation. Pindolol (10^?7M) significantly suppressed the effects evoked by isoprenaline, but did not affect those produced by dopamine. The dopamine receptor antagonist metoclopramide (5×10^?5 and 10^?4M), however, shifted the dose-response curve for dopamine-induced relaxation significantly to the right in a concentration dependent manner without affecting relaxations caused by isoprenaline or papaverine. These results demonstrate for the first time a specific antagonism to dopamine-induced relaxation on rabbit mesenteric arteries $\text{in vitro}$. They support the hypothesis of the existence of specific dopamine receptors in vascular smooth muscles.     

Thr164Ile polymorphism of the human beta2-adrenoceptor exhibits blunted desensitization of cardiac functional responses in vivo

In subjects heterozygous for Thr164Ile beta2-adrenoceptor (beta2AR) polymorphism, cardiac responses to beta2AR agonist stimulation are blunted. In this study, we investigated agonist-induced desensitization of Thr164Ile beta2ARs. For this purpose, we assessed in six subjects with heterozygous Thr164Ile beta2ARs and in 10 subjects with homozygous wild-type (WT) beta2ARs the effects of 2-wk oral treatment with 3 x 5 mg/day terbutaline on terbutaline infusion-induced increases in heart rate (HR) and contractility [measured as shortening of HR-corrected duration of electromechanical systole (QS2c)]. Compared with WT beta2AR subjects, Thr164Ile subjects exhibited a blunted terbutaline-induced maximum increase in HR (WT 32 +/- 4 beats/min, Thr164Ile 19 +/- 3 beats/min, P < 0.05) and contractility (WT -54 +/- 2 ms, Thr164Ile -37 +/- 6 ms, P < 0.05). Two-week oral terbutaline treatment desensitized cardiac beta2AR responses to terbutaline infusion (increase in HR: WT 10 +/- 2 beats/min, Thr164Ile 8 +/- 4 beats/min; increase in contractility: WT -22 +/- 5 ms Thr164Ile: -17 +/- 6 ms); however, the extent of desensitization was larger in WT than Thr164Ile beta2AR subjects. Thus, after 2-wk oral terbutaline treatment cardiac, beta2AR responses did not differ anymore between WT and Thr164Ile beta2AR subjects. We conclude that agonist-induced desensitization of cardiac beta2ARs is more pronounced in WT than Thr164Ile subjects. Thus cardiac Thr164Ile subjects appear to be somewhat protected against agonist-induced desensitization.