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INTRODUCTION: Pupillary function is a sensitive pharmacological readout in humans, but drug effects are not well characterized in rodents. Hence improved methods are needed for quantifying pupillary responses in conscious albino rodents. METHODS: A novel camera-based pupillary imaging method was developed for conscious rats under red-light settings to investigate the effect of various reference compounds on pupil and iris diameter and light reflex. The chosen compounds (pilocarpine, tropicamide, phenylephrine, clonidine, cocaine, morphine) possess well characterized effects in humans. In rats, the compounds were administered topically as eye drops and/or systemically by intraperitoneal injection. RESULTS: Red-light was utilized in order to induce intermediate pupillary width at baseline conditions in conscious albino and pigmented rats, thereby enabling detection of light-induced constriction (miosis) and dilation (mydriasis) of the pupil. Pupil diameter and light reflex were affected by both topical and systemic administration of pilocarpine, tropicamide, phenylephrine, clonidine, cocaine, morphine. However only pharmacologically induced mydriasis was found in albino rats, whereas pigmented rats displayed both mydriasis and miosis albeit with differential responses. The pupillary imaging system is suitable for stand-alone studies as well as for pupil function assessment within the frame of a typical CNS study evaluating behavior (modified Irwin test), locomotor activity and body temperature. DISCUSSION: Strain-specific pharmacological responses were detected for pupillary function, with slightly more human-like responses in pigmented rats than albino rats, illustrating that effects on the rat pupil/iris cannot be directly translated to humans. Nevertheless, changes in pupillary diameter and/or pupillary light reflex in rats is a functional endpoint that can be quantified during early testing, and may trigger more detailed mechanistic characterization beyond the safety pharmacology core CNS battery.
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INTRODUCTION: Irwin/FOB testing is routinely conducted to investigate the neurofunctional integrity of laboratory animals during preclinical development of new drugs, however, the study design frequently varies to meet specific needs. Representatives of several European-based pharmaceutical companies performed a "state-of-the-art" assessment of how they conduct their CNS safety evaluation using Irwin/FOB tests. METHODS: This assessment consisted of (1) a survey of current/historical practice, (2) an evaluation of historical studies with reference compounds (amphetamine, chlorpromazine) to determine intercompany reproducibility of results, and (3) an interlaboratory test using reference compounds (MK-801, chlorpromazine) to determine whether partially standardized conditions (animals, sex, doses, vehicles, administration route, observation time points, systemic exposure) might reduce variability of results. RESULTS: Our survey revealed several similarities, e.g., main endpoints of home cage and openfield observations, species, and positive control substances, but also a high level of heterogeneity between different companies with regard to behavioral endpoints during handling and reflex testing, scoring, group size, and timing of studies. Analysis of heterogeneously designed historical studies with amphetamine and chlorpromazine showed the anticipated behavioral changes, albeit with quantitative variability, and identified more robust (e.g., activity, posture, muscle tone, startle reflex, body temperature) and less robust (piloerection, stereotypical behavior, palpebral closure, respiration) Irwin/FOB parameters. A partially standardized interlaboratory test with MK-801 and chlorpromazine showed the expected behavioral changes and principally confirmed the historically-based more/less robust Irwin/FOB parameters, however, it also showed exposure variability and did not show a markedly reduced quantitative variability of behavioral results. DISCUSSION: Our survey and intercompany test results demonstrate certain heterogeneity in design and conduct of Irwin/FOB tests by pharmaceutical companies. Although the general behavioral profiles for the reference compounds were consistently found, quantitative variability of results remained even under partially standardized conditions. This suggests the importance of a high level of standardization with regard to the Irwin/FOB test modification used, scoring system, and observer training, in order to achieve an improved intercompany comparability of Irwin/FOB results.
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Hydrogenation reactions starting with di- or oligonuclear boron compounds featuring direct B-B bonds with the B atoms in the formal oxidation state +II are analyzed with the aid of ab initio quantum chemical (RI-MP2) calculations. The products of these reactions are B(III) hydrides which might be useful starting reagents for stoichiometric hydrogenation reactions and possibly in special cases also for hydrogen storage. Several different isomers of these B(III) hydrides featuring either terminal or bridging H atoms were considered. The results are compared to hydrogenation reactions of related molecules.
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The first structurally characterized mononuclear biscarbamate complex of Zn with eta1-coordinated carbamate ligands can be prepared by reaction between [EtZn(O2CN(iBu)2)]4 and a guanidine base. Usage of a weaker base leads to eta2-coordinated complexes.
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Carbamatos/química , Metaloproteínas/química , Modelos Químicos , Compuestos Organometálicos/química , Hidrolasas de Triéster Fosfórico/química , Zinc/química , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Electrones , Activación Enzimática , Guanidina/química , Ligandos , Modelos Moleculares , Estructura Molecular , EstereoisomerismoRESUMEN
Dinuclear eta2,micro2-bonded amidinate complexes to group 13 element hydrides are of potential interest for applications in the field of hydrogen storage. In this work repeated dihydrogen elimination starting with amidine-stabilized boron, aluminum, and gallium hydrides is discussed on the basis of quantum chemical calculations which give useful information about the thermodynamic properties of these reactions and the possible reaction pathways in dependence of the chosen amidine derivative. It will be shown that, in agreement to recent experimental work, the thermodynamic properties are greatly influenced by the nature of the substituents bonded to the amidine. The amidine stabilized hydrides first eliminate dihydrogen in an intramolecular process leading to mononuclear amidinate complexes. These complexes could dimerize, if the amidine carries not too bulky organic groups, to give dinuclear complexes featuring two eta2,micro2-coordinated amidinate ligands. Further dihydrogen elimination leads to the generation of a dinuclear species with two group 13 elements (E) in the formal oxidation state +II and direct E-E bonding. Finally, elimination of another H2 for E = B possibly gives amidinate complexes featuring a double bond between two boron atoms in the formal oxidation state +I.
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Absorption spectra were measured for Ti2 in Ne and Ar matrices. The spectra give evidence for several electronic transitions in the region between 4000 and 10 000 cm(-1) and provide important information about some excited electronic states of Ti2 in proximity to the ground state. The vibrational fine structure measured for these transitions allowed to calculate the force constants and the anharmonicity of the potential energy curves of the excited states, and to estimate changes in the internuclear Ti-Ti distances relative to the electronic ground state. The quantum chemical studies confirm the previously suggested (3)Delta(g) state as the ground state of Ti2. The equilibrium bond distance is calculated to be 195.4 pm. The calculated harmonic frequency of 432 cm(-1) is in good agreement with the experimental value of 407.0 cm(-1). With the aid of the calculations it was possible to assign the experimentally observed transitions in the region between 4000 and 10 000 cm(-1) to the 1 (3)Pi(u)<--(3)Delta(g), 1 (3)Phi(u)<--(3)Delta(g), 2 (3)Pi(u)<--(3)Delta(g), 2 (3)Phi(u)<--(3)Delta(g), and (3)Delta(u)<--(3)Delta(g) excitations (in the order of increasing energy). The calculated relative energies and harmonic frequencies are in pleasing agreement with the experimentally obtained values, with deviations of less than 5% and 2%, respectively. The bond distances estimated on the basis of the experimental spectra tally satisfactorily with the predictions of our calculations.
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BACKGROUND: Clinical and experimental evidence suggest that the parasympathetic nervous system is involved in the pathogenesis of atrial fibrillation (AF). However, it is unclear whether changes in G-protein-coupled inward rectifying K(+) current (I(K,ACh)) contribute to chronic AF. METHODS AND RESULTS: In the present study, we used electrophysiological recordings and competitive reverse-transcription polymerase chain reaction to study changes in I(K,ACh) and the level of the I(K,ACh) GIRK4 subunit in isolated human atrial myocytes and the atrial tissue of 39 patients with sinus rhythm and 24 patients with chronic AF. The density of I(K,ACh) was approximately 50% smaller in myocytes from patients with AF compared with those in sinus rhythm, and this was accompanied by decreased levels of GIRK4 mRNA. The current density of the inward rectifying K(+) current (I(K1)) was 2-fold larger during AF than in sinus rhythm, in correspondence with an increase in Kir2.1 mRNA. The larger I(K1) in AF is consistent with more negative membrane potentials in right atrial trabeculae from AF patients. Moreover, action potential duration was reduced in AF, and the action potential shortening produced by muscarinic receptor stimulation was attenuated, indicating that the changes of I(K1) and I(K,ACh) were functionally relevant. CONCLUSIONS: Chronic human AF induces transcriptionally mediated upregulation of I(K1) but downregulation of I(K,ACh) and attenuates the muscarinic receptor-mediated shortening of atrial action potentials. This suggests that atrial myocytes adapt to a chronically high rate by downregulating I(K,ACh) to counteract the shortening of the atrial effective refractory period due to electrical remodeling.
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Potenciales de Acción , Fibrilación Atrial/genética , Fibrilación Atrial/fisiopatología , Canales de Potasio de Rectificación Interna/fisiología , Canales de Potasio/biosíntesis , Receptores Muscarínicos/fisiología , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Anciano , Fibrilación Atrial/metabolismo , Carbacol/farmacología , Células Cultivadas , Enfermedad Crónica , Regulación hacia Abajo , Conductividad Eléctrica , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Atrios Cardíacos/citología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Masculino , Agonistas Muscarínicos/farmacología , Miocardio/citología , Canales de Potasio/genética , ARN Mensajero/biosíntesisRESUMEN
The thiadiazinone derivatives EMD 60417, EMD 66430, and EMD 66398 were developed as class III antiarrhythmic agents. Their chemical structure is closely related to that of their calcium-sensitizing congener [+]-EMD 60263, and EMD 66398 possesses the methylsulfonylaminobenzoyl moiety present in the prototypical IKr blocker E-4031. We compared the electrophysiologic effects of these compounds with standard drugs (almokalant, E-4031, quinidine) in cardiac myocytes from guinea-pig ventricle and human atrium by whole-cell patch-clamp technique. The test compounds' class III action, which is related to impairment of K+ channel function, was confirmed by action potential measurements. EMD 60417, EMD 66430, EMD 66398, and almokalant (1 microM each) reversibly prolonged the action potential duration in guinea-pig myocytes. In the same cells, the rapidly activating component IKr of the delayed rectifier K+ current, which has been defined by its sensitivity to E-4031, was reduced by EMD 60417, EMD 66430, EMD 66398, and almokalant. Inhibition of IKr was concentration-dependent as determined by attenuation of tail currents. The slowly activating component IKs of the delayed rectifier K+ current was not affected. The inward rectifier K+ current IK1 was not influenced at potentials close to the reversal potential. Transient and sustained outward K+ currents (Ito, Iso) measured in human atrial myocytes were not altered by any EMD compound. L-type Ca2+ current was hardly affected at concentrations of 1-10 microM, but sodium current was decreased. Action potential prolongation by EMD 60417, EMD 66430, and EMD 66398 is due to block of IKr. INa is inhibited at higher concentrations by EMD 66430 and EMD 60417. EMD 66398 is more potent and selective for IKr than EMD 60417 and EMD 66430, and thus resembles E-4031 in structure and function.
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Antiarrítmicos/farmacología , Corazón/efectos de los fármacos , Miocardio/metabolismo , Tiadiazinas/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Antiarrítmicos/química , Canales de Calcio/metabolismo , Electrofisiología , Cobayas , Corazón/fisiología , Humanos , Técnicas In Vitro , Estructura Molecular , Miocardio/citología , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Canales de Sodio/metabolismo , Tiadiazinas/químicaRESUMEN
Thermal decomposition of monochlorogallane, [H2GaCl]n, at ambient temperatures releases H2 and results in the formation of gallium(I) species, including the new compound Ga[GaHCl3], which has been characterized crystallographically at 100 K (monoclinic P2(1)/n, a = 5.730(1), b = 6.787(1), c = 14.508(1) A, beta = 97.902(5) degrees ) and by its Raman spectrum. The gallane suffers symmetrical cleavage of the Ga(mu-Cl)2Ga bridge in its reaction with NMe3 but unsymmetrical cleavage, giving [H2Ga(NH3)2](+)Cl(-), in its reaction with NH3. Ethene inserts into the Ga-H bonds to form first [Et(H)GaCl]2 and then [Et2GaCl]2.
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Beneficial therapeutic effects of dihydropyridine derivatives in cardiovascular and neurological disorders are often associated with selective L-type Ca(2+)channel blockade. Here the new dihydropyridine derivatives Bay E5759 (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid ethyl-1-methylethyl ester) and Bay A4339 (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl-ester) were tested for their potency and selectivity of blocking of Ba(2+)currents mediated by low-(LVACC)vs high-voltage activated Ca(2+)channels (HVACC) in neuroblastoma-glioma hybrid cells. Nisoldipine and mibefradil served as reference compounds. Bay E5759 and Bay A4339 blocked HVACC at low nanomolar concentrations, whereas LVACC was hardly reduced at up to 10 microM. The order of potency for blockade of HVACC was Bay E5759 (IC(50): 0.4 nM) > Bay A4339 (2.5 nM) approximately = nisoldipine (4 nM) >> mibefradil (3.8 microM). Thus Bay E5759 and Bay A4339 are highly potent and selective blockers of HVACC, presumably L-type Ca(2+)channels.
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Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Nifedipino/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electrofisiología , Células Híbridas , Mibefradil/farmacología , Ratones , Nifedipino/análogos & derivados , Nisoldipino/farmacología , Ratas , Células Tumorales CultivadasRESUMEN
Ascorbic acid appears to have vasodilatory properties, but the underlying mechanisms are not well understood. The aims of this study were to define the acute effects of locally infused ascorbic acid in human veins and to explore underlying mechanisms by using pharmacological tools in vivo. Ascorbic acid was infused in dorsal hand veins submaximally preconstricted with the alpha(1)-adrenoceptor agonist phenylephrine or with prostaglandin F(2alpha) in 23 healthy male nonsmokers, and the venodilator response was measured. Ascorbic acid produced dose-dependent dilation with maximum reversal of constriction of 38+/-4% in phenylephrine-preconstricted veins and of 51+/-13% in prostaglandin F(2alpha)-preconstricted veins. Oral pretreatment with the cyclooxygenase inhibitor acetylsalicylic acid or local coinfusion of ascorbic acid and the nitric oxide synthase inhibitor N:(G)-monomethyl-L-arginine had no effect, but coinfusion of ascorbic acid and methylene blue (to inhibit cGMP generation) abolished venodilation. Coinfusion of ascorbic acid and the nonselective potassium channel blocker quinidine abolished venodilation, whereas the inhibitor of ATP-dependent potassium channels glibenclamide had no effect. In cultured bovine endothelial cells, ascorbic acid did not affect intracellular calcium concentration but blunted the response to ATP or digitonin exposure. Ascorbic acid, in millimolar concentrations, dilates human hand veins, presumably by activation of vascular smooth muscle potassium channels through cGMP. This activation is independent of eNOS-mediated nitric oxide synthesis and cyclooxygenase products and does not involve ATP-dependent potassium channels.
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Ácido Ascórbico/farmacología , Músculo Liso Vascular/efectos de los fármacos , Venas/efectos de los fármacos , Adulto , Animales , Aspirina/farmacología , Calcio/metabolismo , Bovinos , Células Cultivadas , Inhibidores de la Ciclooxigenasa/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Mano , Humanos , Infusiones Intravenosas , Masculino , Azul de Metileno/farmacología , Músculo Liso Vascular/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fenilefrina , Canales de Potasio/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Venas/fisiología , omega-N-Metilarginina/farmacologíaRESUMEN
Upon deposition of Al, Ga, or In atoms (M) together with phosphine in a solid argon matrix, metal atom complexes M.PH3 are formed. Photolysis of the matrices at lambda = 436 nm results in the tautomerization of the adduct species to the insertion products HMPH2 and H2MPH. In addition, PH is formed from the reactions with Ga and In, with HMPH2 being its most likely precursor. Further photolysis into the absorption maximum of HMPH2 near 550 nm results in decomposition of HMPH2 with partial re-formation of the adduct M.PH3 and further buildup of PH. H2MPH is photostable under these conditions but suffers decomposition under the action of UV light (200 < or = lambda < or = 400 nm). All the molecules have been identified by their IR spectra, the assignments being attested by the effects of deuteration and also by comparison either with the vibrational properties anticipated by density functional theory (DFT) calculations or with those of known, related molecules. The resulting analysis is elaborated for the light it sheds on the structures and properties of the new molecules and on the mechanisms of the reactions affording, or disposing of, them.
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In most macrovascular endothelial cell (EC) preparations, resting membrane potential is determined by the inwardly rectifying K+ current (I(K1)), whereas in microvascular EC the presence of I(K1) varies markedly. Cultured microvascular EC from small vessels of human omentum were examined by means of the voltage-clamp technique to elucidate the putative role of I(K1) in maintaining resting membrane potential. Macrovascular EC from human iliac artery and bovine aorta served as reference. Human omentum EC showed an outwardly rectifying current-voltage relation. Inward current was hardly sensitive to variations of extracellular [K+] and Ba2+ block suggesting lack of I(K1). However, substitution of extracellular [Na+] and/or [Cl-] affected the current-voltage relation indicating that Na+ and Cl- contribute to basal current. Furthermore, outward current was reduced by tetraethylammonium (10 mM), and cell-attached recordings suggested the presence of a Ca2+-activated K+ current. In contrast to human omentum EC, EC from human iliac artery and bovine aorta possessed inwardly rectifying currents which were sensitive to variations of extracellular [K+] and blocked by Ba2+. Thus, the lack of I(K1) in human omentum EC suggests that resting membrane potential is determined by Na+ and Cl- currents in addition to K+ outward currents.
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Endotelio Vascular/citología , Endotelio Vascular/fisiología , Epiplón/irrigación sanguínea , Canales de Potasio de Rectificación Interna/fisiología , Animales , Aorta/citología , Bario/farmacología , Bovinos , Células Cultivadas , Cloruros/farmacocinética , Humanos , Arteria Ilíaca/citología , Potenciales de la Membrana/fisiología , Microcirculación/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Sodio/farmacocinéticaRESUMEN
BACKGROUND: A C825T polymorphism was recently identified in the human gene encoding for the beta(3)-subunit of heterotrimeric G proteins. The 825T allele is associated with a splice variant of Gbeta(3) and enhanced signal transduction. We hypothesized that patients carrying the 825T allele exhibit the modified Gbeta(3) phenotype. The resulting enhancement of signal transduction should be detectable in the Gbetagamma-dimer-mediated acetylcholine-stimulated K(+) current (I(K,ACh)). METHODS AND RESULTS: Seventy patients undergoing cardiac surgery were genotyped for the C825T polymorphism. In right atrial myocytes from these patients, the inward rectifier K(+) currents (I(K1), I(K,ACh)) were studied with the whole-cell patch-clamp technique. Background current I(K1) was measured with depolarizing ramp pulses and quantified as inward current at -100 mV; mean amplitudes were (pA/pF) 4.98+/-0.49 (n=30/93 patients/cells) in patients with CC genotype, 4.25+/-0.36 (n=31/121 patients/cells) with TC, and 7. 46+/-1.14 (n=9/32 patients/cells; P<0.05) with TT. Conversely, mean I(K,ACh), which is maximally activated by carbachol (2 micromol/L), was reduced in patients with TT genotype (pA/pF, 4.30+/-1.33, n=9/27 patients/cells; P<0.05) compared with the other 2 groups (6.56+/-0. 54, n=30/80 and 6.16+/-0.45, n=31/117 patients/cells, for CC and TC genotype, respectively). Essentially similar results were obtained with adenosine (1 mmol/L). CONCLUSIONS: We found an association between the Gbeta(3) 825T allele and amplitude of human atrial I(K1) and I(K,ACh). Increased background current density in TT carriers could shorten action potential duration and may be due to I(K,ACh) being constitutively active in this genotype.
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Alelos , Proteínas de Unión al GTP/genética , Corazón/fisiopatología , Canales de Potasio de Rectificación Interna , Canales de Potasio/fisiología , Anciano , Bloqueadores de los Canales de Calcio/uso terapéutico , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Femenino , Genotipo , Corazón/efectos de los fármacos , Atrios Cardíacos , Cardiopatías/tratamiento farmacológico , Cardiopatías/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Miocardio/patología , Polimorfismo Genético/genéticaRESUMEN
Sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) have been reported to activate muscarinic receptor-activated inward rectifier K(+) current (I(K.ACh)) in cultured guinea pig atrial myocytes with similar nanomolar potency. Members of the endothelial differentiation gene (Edg) receptor family were recently identified as receptors for SPP; however, these receptors respond only to micromolar concentrations of SPPC. Here we investigated the sphingolipid-induced activation of I(K.ACh) in freshly isolated guinea pig, mouse, and human atrial myocytes. SPP activated I(K.ACh) in atrial myocytes from all three species with a similar nanomolar potency (EC(50) values: 4-8 nM). At these low concentrations, SPPC also activated I(K.ACh) in guinea pig myocytes. In contrast, SPPC was almost ineffective in mouse and human myocytes, thus resembling the pharmacology of the Edg receptors. Transcripts of Edg-1, Edg-3, and Edg-5 were detected in human atrial cells. Moreover, activation of I(K.ACh) by SPP was blocked by the Edg-3-selective antagonist suramin, which did not affect basal or carbachol-stimulated K(+) currents. In conclusion, these data indicate that I(K.ACh) activation by SPP and SPPC exhibits large species differences. Furthermore, they suggest that SPP-induced I(K.ACh) activation in human atrial myocytes is mediated by the Edg-3 subtype of SPP receptors.
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Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Lisofosfolípidos , Miocardio/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacología , Análisis de Varianza , Animales , Femenino , Cobayas , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa , Receptores Lisofosfolípidos , Especificidad de la EspecieRESUMEN
OBJECTIVE: Amiodarone, a class III antiarrhythmic agent, is a potent vasodilator in vivo. Its main metabolite, N-desethylamiodarone, contributes to the antiarrhythmic action of amiodarone after long-term treatment. It is unknown whether N-desethylamiodarone has acute vascular effects. The aim of this study was to explore the mechanism of action of N-desethylamiodarone in human hand veins. METHODS: The dorsal hand vein compliance technique was applied in 36 healthy male volunteers. In hand veins preconstricted with the alpha1-adrenergic receptor agonist phenylephrine or prostaglandin F2alpha, N-desethylamiodarone and an inhibitor of nitric oxide formation (N(G)-monomethyl-L-arginine, L-NMMA) were infused in the presence or absence of a cyclooxygenase inhibitor (acetylsalicylic acid), and the venodilator effect was measured. Furthermore, N-desethylamiodarone was infused after oral treatment with hydrocortisone or coinfused with alpha-tocopherol. Additional experiments were carried out in bovine aortic endothelial cells to explore the effects of N-desethylamiodarone on the intracellular Ca2+ concentration ([Ca2+]i). RESULTS: N-Desethylamiodarone produced dose-dependent venodilation (47% +/- 4% maximum). In vitro, 10 micromol/L N-desethylamiodarone caused a sustained increase of the endothelial [Ca2+]i. Pretreatment of the volunteers with acetylsalicylic acid reduced the maximum N-desethylamiodarone-induced venodilation to 22% +/- 8%; L-NMMA reduced the maximum N-desethylamiodarone-induced venodilation to 18% +/- 11%. Pretreatment with acetylsalicylic acid and coinfusion of N-desethylamiodarone and L-NMMA abolished the venodilation, whereas hydrocortisone had no effect. Coinfusion of alpha-tocopherol and N-desethylamiodarone reduced the maximum N-desethylamiodarone-induced venodilation to 11% +/- 4%. CONCLUSIONS: In concentrations estimated to be in the therapeutic range, N-desethylamiodarone dilates preconstricted human hand veins in vivo and increases endothelial [Ca2+]i in vitro. Subsequently the cyclooxygenase (COX-1) and the endothelial nitric oxide synthase pathways are activated. The resulting venodilation does not involve inflammatory cytokines, inducible nitric oxide synthase, or inducible cyclooxygenase (COX-2).
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Amiodarona/análogos & derivados , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Venas/efectos de los fármacos , Adulto , Amiodarona/administración & dosificación , Amiodarona/farmacología , Animales , Aorta , Presión Sanguínea/efectos de los fármacos , Calcio/metabolismo , Bovinos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Mano/irrigación sanguínea , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Infusiones Intravenosas , Masculino , Valores de Referencia , Factores de Tiempo , Vasodilatadores/administración & dosificaciónRESUMEN
The main in-vivo metabolite of amiodarone, N-desethylamiodarone (DEAM), possesses clinically relevant class-II antiarrhythmic and vasodilator activities. Vasodilation by DEAM is endothelium dependent and involves a sustained and biphasic increase in cytosolic free Ca2+ concentration ([Ca2+]i). The aims of this study were to explore the mechanisms mediating the DEAM-induced increase in [Ca2+]i in endothelial cells and to determine whether this increase in [Ca2+]i was associated with altered cell proliferation. Cultured bovine aortic endothelial cells were loaded with the Ca2+-sensitive fluorescent dye Fura-2/AM, and [Ca2+]i measured spectrofluorimetrically. DEAM increased [Ca2+]i concentration dependently (EC50 approximately 6 microM) both in the presence and absence of extracellular Ca2+. In the presence of extracellular Ca2+, the response of [Ca2+]i to DEAM (10 microM) consisted of an initial rise to a plateau followed by a second increase to micromolar levels. The initial plateau was reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (200 nM) and by the antioxidant ascorbic acid (100 microM). The initial rate of rise in [Ca2+]i was decreased by blocking mitochondrial Ca2+ release with cyclosporine A (1 microM). Under Ca2+-free conditions, the response of [Ca2+]i to DEAM (10 microM) was also biphasic, consisting of an initial transient peak and a second slow increase. When extracellular Ca2+ was restored, [Ca2+]i rose to micromolar concentrations. The initial peak was abolished by thapsigargin, but not altered by ascorbic acid or cyclosporine A. Both the second [Ca2+]i increase and that due to restoring extracellular Ca2+ were reduced by ascorbic acid but not affected by thapsigargin or cyclosporine A. The DEAM-induced generation of free radicals and sustained increase in [Ca2+]i might alter cell proliferation and endothelial cell proliferation was indeed concentration-dependently inhibited by DEAM (IC50 approximately 2.5 microM). In conclusion, the DEAM-induced [Ca2+]i increase in endothelial cells is due to Ca2+ influx from the extracellular space and to Ca2+ release from endoplasmic reticulum and mitochondria and involves enhanced generation of free radicals.
Asunto(s)
Amiodarona/farmacología , Calcio/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Amiodarona/análogos & derivados , Animales , Antiarrítmicos/farmacología , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Calcio/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/metabolismo , Homeostasis/efectos de los fármacos , Cinética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Tapsigargina/farmacología , Vasodilatadores/farmacologíaRESUMEN
OBJECTIVES: The reported increase in basal activity of hearts from transgenic mice (TG4) overexpressing the human beta 2-adrenoceptor (beta 2-AR) was explained by spontaneously active beta 2-ARs that stimulate the beta-adrenergic cascade in the absence of an agonist. In order to examine altered myocardial function on a cellular level, we have investigated L-type calcium current (ICa,L) and cell shortening in ventricular myocytes from TG4 hearts. Myocytes from littermates (LM) and wild type animals (WT) served as controls. METHODS: Cardiac beta-AR density was measured by [125I]-iodocyanopindolol binding to ventricular membranes. ICa,L was assessed by standard whole-cell voltage clamp technique. Contractility was measured as cell shortening in ventricular myocytes and as force of contraction in electrically stimulated left atria. RESULTS: Overexpression of beta 2-ARs was confirmed by an almost 400-fold increase in beta-AR density. The beta 1:beta 2-AR ratio in WT mice was 71:29. Myocytes from TG4 and LM mice were similar in size as judged by membrane capacitance and two dimensional cell area. ICa,L amplitude was significantly lower in TG4 than in LM myocytes (with 2 mM [Ca2+]o -4.82 +/- 0.48 vs. -6.56 +/- 0.38 pA/pF, respectively). In TG4 myocytes, the ICa,L response to isoproterenol (1 microM) was almost abolished. Cell shortening was not different in physiological [Ca2+]o, but smaller in maximum [Ca2+]o when comparing TG4 to control myocytes. Basal force of contraction in left atria did not differ between TG4 and LM at any age investigated. In TG4 left atria the inotropic response to isoproterenol was also absent, whereas responses to high [Ca2+]o or dibutyryl-cAMP (1 mM) were present but reduced. The rate of spontaneous beating of right atria was elevated in TG4 mice. CONCLUSIONS: Since only spontaneous beating rate but neither basal ICa,L amplitude nor basal contractile activity were elevated, our data fail to reveal evidence for spontaneously active, stimulating beta 2-ARs in left atrium and ventricle. A contractile deficit unrelated to the beta-adrenoceptor pathway is evident in TG4 myocytes and left atria.
Asunto(s)
Canales de Calcio/metabolismo , Contracción Miocárdica , Miocardio/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacología , Análisis de Varianza , Animales , Bucladesina/farmacología , Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Tamaño de la Célula , Células Cultivadas , Estimulación Eléctrica , Expresión Génica , Atrios Cardíacos , Yodocianopindolol/farmacología , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Miocardio/citología , Técnicas de Placa-Clamp , Receptores Adrenérgicos beta 2/genética , Transducción de Señal , Estadísticas no Paramétricas , Estimulación QuímicaRESUMEN
In rat ventricle, two Ca(2+)-insensitive components of K(+) current have been distinguished kinetically and pharmacologically, the transient, 4-aminopyridine (4-AP)-sensitive I(to) and the sustained, tetraethylammonium (TEA)-sensitive I(K). However, a much greater diversity of depolarization-activated K(+) channels has been reported on the level of mRNA and protein. In the search for electrophysiological evidence of further current components, the whole cell voltage-clamp technique was used to analyze steady-state inactivation of outward currents by conditioning potentials in a wide voltage range. Peak (I(peak)) and late (I(late)) currents during the test pulse were analyzed by Boltzmann curve fitting, producing three fractions each. Fractions a and b had different potentials of half-maximum inactivation (V(0.5)); the third residual fraction, r, did not inactivate. Fractions a for I(peak) and I(late) had similar relative amplitudes and V(0.5) values, whereas size and V(0.5) of fractions b differed significantly between I(peak) and I(late). Only b of I(peak) was transient, suggesting a relation with I(to), whereas a, b, and r of I(late) appeared to be three different sustained currents. Therefore, four individual outward current components were distinguished: I(to) (b of I(peak)), I(K) (a), the steady-state current I(ss) (r), and the novel current I(Kx) (b of I(late)). This was further supported by differential sensitivity to TEA, 4-AP, clofilium, quinidine, dendrotoxin, heteropodatoxin, and hanatoxin. With the exception of I(to), none of the currents exhibited a marked transmural gradient. Availability of I(K) was low at resting potential; nevertheless, I(K) contributed to action potential shortening in hyperpolarized subendocardial myocytes. In conclusion, on the basis of electrophysiological and pharmacological evidence, at least four components contribute to outward current in rat ventricular myocytes.
Asunto(s)
Potenciales de Acción , Ventrículos Cardíacos/citología , Función Ventricular , 4-Aminopiridina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Células Cultivadas , Venenos Elapídicos/farmacología , Electrofisiología , Ventrículos Cardíacos/efectos de los fármacos , Masculino , Neurotoxinas/farmacología , Técnicas de Placa-Clamp , Péptidos/farmacología , Bloqueadores de los Canales de Potasio , Ratas , Ratas Wistar , Tetraetilamonio/farmacologíaRESUMEN
Cardiac intracellular Na+and Ca2+homeostasis is regulated by the concerted action of ion channels, pumps and exchangers. The Na+, K+-ATPase produces the electrochemical concentration gradient for Na+, which is the driving force for Ca2+removal from the cytosol via the Na+/Ca2+exchange. Reduction of this gradient by increased intracellular Na+concentration leads to cellular Ca2+overload resulting in arrhythmias and contractile dysfunction. Na+and Ca2+overload-associated arrhythmias can be produced experimentally by inhibition of Na+efflux (digitalis-induced intoxication) and by abnormal Na+influx via modulated Na+channels (veratridine, DPI 201-106; hypoxia) or via the Na+, H+exchanger. Theoretically, blockers of Na+and Ca2+channels, inhibitors of abnormal oscillatory release of Ca2+from internal stores or modulators of the Na+, Ca2+and Na+, H+exchanger activities could protect against cellular Na+and Ca2+overload. Three exemplary drugs that prevent Na+and Ca2+overload, i.e. the benzothiazolamine R56865, the methylenephenoxydioxy-derivative CP-060S, and the benzoyl-guanidine Hoe 642, a Na+, H+exchange blocker, are briefly reviewed with respect to their efficacy on digitalis-, veratridine- and ischaemia/reperfusion-induced arrhythmias.