RESUMO
ArtinM is a D-mannose binding lectin that has been arousing increasing interest because of its biomedical properties, especially those involving the stimulation of Th1 immune response, which confers protection against intracellular pathogens. The potential pharmaceutical applications of ArtinM have motivated the production of its recombinant form (rArtinM) so that it is important to compare the sugar-binding properties of jArtinM and rArtinM in order to take better advantage of the potential applications of the recombinant lectin. In this work, a biosensor framework based on a Quartz Crystal Microbalance was established with the purpose of making a comparative study of the activity of native and recombinant ArtinM protein. The QCM transducer was strategically functionalized to use a simple model of protein binding kinetics. This approach allowed for the determination of the binding/dissociation kinetics rate and affinity equilibrium constant of both forms of ArtinM with horseradish peroxidase glycoprotein (HRP), a N-glycosylated protein that contains the trimannoside Manα1-3[Manα1-6]Man, which is a known ligand for jArtinM (Jeyaprakash et al., 2004). Monitoring of the real-time binding of rArtinM shows that it was able to bind HRP, leading to an analytical curve similar to that of jArtinM, with statistically equivalent kinetic rates and affinity equilibrium constants for both forms of ArtinM. The lower reactivity of rArtinM with HRP than jArtinM was considered to be due to a difference in the number of Carbohydrate Recognition Domains (CRDs) per molecule of each lectin form rather than to a difference in the energy of binding per CRD of each lectin form.
Assuntos
Técnicas Biossensoriais/instrumentação , Glicoproteínas/química , Peroxidase do Rábano Silvestre/química , Lectina de Ligação a Manose/química , Sistemas Microeletromecânicos/instrumentação , Mapeamento de Interação de Proteínas/instrumentação , Sistemas Computacionais , Desenho de Equipamento , Análise de Falha de Equipamento , CinéticaRESUMO
BACKGROUND: The deficiency of Rh proteins on red blood cells (RBCs) from individuals of the Rh(null) amorph type are the result of homozygosity for a silent RHCE in cis with a deleted RHD. A novel mutation in RHce was identified in two Caucasian Brazilian girls with the amorph type of Rh(null) who were born to parents who were first cousins. STUDY DESIGN AND METHODS: RBCs from the Rh(null) sisters and from family members were analyzed by serology and flow cytometry with specific antibodies. Genomic DNA and transcripts were tested by polymerase chain reaction and sequence analysis. RESULTS: Rh(null) RBCs were nonreactive with anti-Rh and anti-LW. Molecular analyses showed a deletion of RHD and of one nucleotide (960/963; GGGG-->GGG) in exon 7 of the RHce. This deletion introduced a frameshift after Gly321, a new C-terminal sequence, and a premature stop codon, resulting in a shorter predicted protein with 357 amino acids. CONCLUSION: The detection of a unique RHce transcript indicated that the two sisters were homozygous, whereas the other family members were heterozygous for the mutation. A novel mutation resulting in the amorph Rh(null) with loss of Rh antigen expression is described.
Assuntos
Mutação , Sistema do Grupo Sanguíneo Rh-Hr/genética , Adulto , Sequência de Bases , Códon de Terminação , Eritrócitos/imunologia , Eritrócitos/metabolismo , Éxons , Feminino , Mutação da Fase de Leitura , Deleção de Genes , Guanina , Homozigoto , Humanos , Dados de Sequência Molecular , Linhagem , Sistema do Grupo Sanguíneo Rh-Hr/sangue , Síndrome , Transcrição GênicaRESUMO
The completion of the first draft of the human genome has provided an unprecedented opportunity to understand the genetic and molecular basis of disease. Parallel developments of new biological technologies, such as transcript profiling, allow scientists to examine almost any biological system in high molecular resolution. Contemporary drug discovery research is now focusing on the identification and validation of pharmaceutical targets in the molecular pathways/systems embedded in this information. Novel therapeutic interventions are being developed and evaluated as a result of this research which will be the basis of innovative pharmaceuticals of the future.
Assuntos
Desenho de Fármacos , Indústria Farmacêutica/tendências , Genética , Pesquisa/tendências , Animais , Biotecnologia , Estudos de Avaliação como Assunto , Genoma Humano , HumanosRESUMO
Dephosphorylation of substrates for cyclic AMP-dependent protein kinase is essential for reversing the effects of this enzyme. It has been proposed that the relevant phosphatase(s) is stimulated by muscarinic cholinergic agonists, thereby accentuating cholinergic antagonism of beta-adrenergic agonists in the heart. To test this hypothesis, dephosphorylation of the three major substrates of cardiac cyclic AMP-dependent protein kinase (phospholamban, troponin-I, and C-protein) was examined. In isolated myocytes, isoproterenol caused concentration-dependent phosphorylation of these three proteins. Simultaneous exposure to acetylcholine with the isoproterenol caused a rightward shift in the concentration-response curve that was similar for protein phosphorylation in myocytes and for the inotropic response of the intact heart. The addition of propranolol after exposure to isoproterenol resulted in protein dephosphorylation, the onset of which was accelerated by acetylcholine. However, acetylcholine did not affect the rate of dephosphorylation for any of the proteins, indicating that phosphatase activity in cardiac muscle is not enhanced by acetylcholine.
Assuntos
Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Síndrome de Abstinência a Substâncias/metabolismo , Acetilcolina/farmacologia , Animais , Proteínas de Ligação ao Cálcio , Cardiotônicos/farmacologia , Proteínas de Transporte , Feminino , Cobaias , Masculino , Proteínas Musculares , Miocárdio/metabolismo , Fosforilação , Troponina I/metabolismoRESUMO
In intact guinea pig ventricles, acetylcholine (ACH) has been shown to attenuate the positive inotropic effects of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, by reducing protein phosphorylation without altering cAMP levels. In the present study, we tested the hypothesis that the cAMP-independent inhibitory action of ACH is also evident in isolated cardiomyocytes. cAMP-dependent protein kinase (PKA) activity ratio (-cAMP/+cAMP) and phosphorylation of phospholamban (PLB) were determined in unlabeled and 32P-labeled guinea pig ventricular cardiomyocytes, respectively. IBMX increased PKA activity ratio and phosphorylation of PLB in a dose-dependent manner. When cardiomyocytes were incubated simultaneously with IBMX (0-1 mM) and ACH (2 microM), ACH attenuated PLB phosphorylation stimulated by low concentration (1O-100 microM) but not by high concentrations (> 200 microM) of IBMX. EC50 value for IBMX-induced phosphorylation of PLB was 32 +/- 6 microM and increased nearly 3-fold after addition of ACH while PKA activity ratio remained unchanged. The rank order of cyclic nucleotide derivatives to phosphorylate PLB was 8 bromo-cAMP > dibutyryl cAMP > 8 bromo-cGMP > dibutyryl cGMP. ACH reduced phosphorylation of PLB stimulated by 8 bromo-cAMP. We conclude that in isolated cardiomyocytes (1) ACH inhibits phosphorylation of PLB stimulated by either IBMX or 8 bromo-cAMP and (2) ACH does not lower IBMX-stimulated PKA activity ratio. These effects of ACH on PLB phosphorylation cannot be explained by a reduction in IBMX-stimulated cAMP levels but may involve the activation of protein phosphatases.
Assuntos
Acetilcolina/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , AMP Cíclico/metabolismo , Miocárdio/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Receptores Muscarínicos/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Bucladesina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cobaias , Ventrículos do Coração , Miocárdio/citologia , Miocárdio/enzimologia , Nucleotídeos Cíclicos/metabolismo , FosforilaçãoRESUMO
Protein phosphatase inhibitor-1 (PPI-1) has been shown to be present in heart tissue and smooth muscle. Whether PPI-1 is present in cardiomyocytes is not known. The purpose of this study was to determine whether PPI-1 is present and is hormonally regulated in cardiomyocytes. A trichloroacetic acid (TCA) extract enriched in PPI-1 was isolated from guinea pig ventricular cardiomyocytes. The TCA extract inhibited the activity of type 1 protein phosphatase by 20 +/- 4% (n = 3 expts). On phosphorylation by the catalytic subunit of adenosine 3', 5'-cyclic monophosphate-dependent protein kinase, the extent of this inhibition was augmented to 4.5-fold. Dephosphorylation of the phosphorylated TCA extract by type 2 protein phosphatase reduced inhibition to 2 +/- 0.2% (n = 3 expts). To determine whether isoproterenol increases phosphorylation of PPI-1 in cardiomyocytes, the TCA extracts were prepared from cardiomyocytes treated with 1 microM isoproterenol and from untreated cardiomyocytes. The inhibitory activity of the TCA extract in untreated cardiomyocytes was 25 +/- 3% (n = 3 expts) and increased to 75 +/- 2% (n = 3 expts) in isoproterenol-treated cardiomyocytes. With the use of a rabbit skeletal muscle PPI-1 antibody, immunoblots of the TCA extract of cardiomyocytes identified a 28-kDa protein. A 28-kDa protein was also immunoprecipitated from a TCA extract isolated from isoproterenol-treated 32P-labeled cardiomyocytes. The immunoprecipitation was blocked by the addition of excess amounts of purified rabbit skeletal muscle PPI-1. Isoproterenol-treated cardiomyocytes increased the phosphorylation of the 28-kDa protein by 232 +/- 20% (n = 3 expts) compared with untreated cardiomyocytes. We conclude that 1) the 28-kDa protein is PPI-1, 2) PPI-1 is present in ventricular cardiomyocytes, and 3) PPI-1 is hormonally regulated. A decrease in type 1 protein phosphatase activity through phosphorylation of PPI-1 may be an important pathway for augmenting cardiac contractility.
Assuntos
Proteínas de Transporte , Peptídeos e Proteínas de Sinalização Intracelular , Isoproterenol/farmacologia , Miocárdio/metabolismo , Proteínas/metabolismo , Animais , Western Blotting , Feminino , Cobaias , Ventrículos do Coração , Masculino , Miocárdio/citologia , Fosforilação , Testes de Precipitina , CoelhosRESUMO
The influence of the adenosine derivative (--)-N6-phenylisopropyladenosine (R-PIA, 1 micron) and 5'N-ethylcarbox-amidoadenosine (NECA, 1 micron) on beta-adrenergic stimulated (isoproterenol, 10 nM) phosphorylation of sarcolemmal (15 kDa protein), sarcoplasmic reticular (phospholamban) and myofibrillar proteins (troponin I, C-protein) was studied in isolated 32P-labeled guinea-pig ventricles. The identification of the 15 kDa protein, phospholamban, troponin I and C-protein was based on their reaction with specific antibodies. Isoproterenol increased contractile parameters (developed tension, rate of tension development, rate of relaxation) and stimulated the phosphorylation state of a 15 kDa protein (now named phospholemman), of phospholamban, troponin I and C-protein (regarded as regulatory proteins). Isoproterenol concomitantly increased myocardial cyclic AMP levels. R-PIA and NECA attenuated the effects of isoproterenol on contractile parameters as well as on the phosphorylation of the regulatory proteins without affecting cyclic AMP levels. The effects of 1 microM R-PIA and 1 microM NECA on the isoproterenol-stimulated phosphorylation of regulatory proteins were blocked by the adenosine receptor antagonist 1.3-dipropyl-8-cyclopentylxanthine (DPCPX, 1 microM). Therefore, it is concluded that adenosine derivatives acting via adenosine receptors can reduce the isoproterenol-stimulated phosphorylation state of the following regulatory proteins: phospholemman, phospholamban, troponin I and C-protein.
Assuntos
Adenosina/análogos & derivados , Agonistas Adrenérgicos beta/farmacologia , Isoproterenol/farmacologia , Miocárdio/metabolismo , Fenilisopropiladenosina/farmacologia , Fosfoproteínas/metabolismo , Agonistas do Receptor Purinérgico P1 , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida) , Animais , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Feminino , Cobaias , Ventrículos do Coração , Masculino , Contração Miocárdica/efeitos dos fármacos , Fosfoproteínas/isolamento & purificação , Radioisótopos de Fósforo , Fosforilação , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Troponina/isolamento & purificação , Troponina/metabolismo , Troponina IRESUMO
An 125I-iodinated ryanodine analog, modified by attaching an iodo-Cbz-beta-alanyl group to the C10eq hydroxy of ryanodine (iodo-carbobenzyloxy-beta-alanyl-ryanodine), binds to cardiac sarcoplasmic reticulum Ca2+ release channels with equal affinity as [3H]ryanodine. In the present study, both iodo-Cbz-beta-alanyl-ryanodine and ryanodine bound to canine cardiac microsomal membrane preparations in a Ca2+ dependent manner. At 10 microM free Ca2+ doxorubicin increased specific binding of both ligands, with doxorubicin concentrations of 4.06 +/- 0.44 and 6.22 +/- 1.31 microM inducing 50% maximal enhancement of binding for ryanodine and iodo-Cbz-beta-alanyl-ryanodine, respectively. Effects of ryanodine and iodo-Cbz-beta-alanyl-ryanodine +/- doxorubicin in vitro on cardiac sarcoplasmic reticulum Ca2+ release were compared indirectly by determining Ca2+ accumulation in cardiac microsomal vesicles loaded with 45Ca2+. In the absence of oxalate, neither ryanodine nor iodo-Cbz-beta-alanyl-ryanodine (10 microM) decreased net Ca2+ uptake, whereas doxorubicin reduced Ca2+ accumulation 20 +/- 2%. In the presence of oxalate and 0.4 microM free Ca2+ ("low"), both ryanodine and iodo-Cbz-beta-alanyl-ryanodine modestly decreased (by 19% and 17% at 10 nM, respectively) maximum Ca2+ accumulation. Increasing concentrations of ryanodine (100 nM-100 microM) and iodo-Cbz-beta-alanyl-ryanodine (100 nM-30 microM) had no greater effect, but 100 microM iodo-Cbz-beta-alanyl- ryanodine decreased net Ca2+ uptake 57 +/- 3%. Doxorubicin (30 microM) alone reduced Ca2+ uptake 36%; its effects with 1 nM-10 microM ryanodine or 1 nM-100 microM iodo-Cbz-beta-alanyl-ryanodine were additive.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cálcio/metabolismo , Doxorrubicina/farmacologia , Miocárdio/metabolismo , Rianodina/análogos & derivados , Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Radioisótopos de Cálcio , Cães , Coração/efeitos dos fármacos , Técnicas In Vitro , Radioisótopos do Iodo , Ligantes , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Ensaio Radioligante , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
Acetylcholine acting via muscarinic cholinoceptors decreased phosphorylation of phospholamban and troponin I without reducing adenosine 3',5'-cyclic monophosphate (cAMP) levels or cAMP-dependent protein kinase activity ratio in the presence of 10-100 nM isoproterenol in guinea pig ventricular myocytes. The effect of acetylcholine was more pronounced when adenosine deaminase (5 U/ml) was present and incubation period was short (10 s). Okadaic acid, an inhibitor of protein phosphatase activity, blocked the acetylcholine-mediated inhibition of isoproterenol-stimulated phosphorylation of phospholamban. It is suggested that acetylcholine reduces protein phosphorylation by a cAMP-independent mechanism in guinea pig ventricular myocytes.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Miocárdio/metabolismo , Receptores Muscarínicos/fisiologia , Troponina/metabolismo , Acetilcolina/farmacologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Éteres Cíclicos/farmacologia , Feminino , Cobaias , Masculino , Nucleotídeos Cíclicos/metabolismo , Ácido Okadáico , Parassimpatomiméticos/farmacologia , Fosforilação/efeitos dos fármacos , Troponina IRESUMO
Prior studies have shown that in vivo systemic administration of pertussis toxin in conscious dogs catalyzes ADP ribosylation of Gi and G(o) proteins, which attenuates intracellular transduction of muscarinic receptor activation. We tested the hypothesis that this impairment may result in the alteration of the following indexes of cardiac vagal activity: baroreflex sensitivity and heart rate variability. Heart rates during submaximal exercise were also determined. These variables were measured in eight conscious dogs before and 72 hr after pertussis toxin administration. Pertussis toxin significantly reduced (P < 0.001) baroreflex sensitivity (from 18.7 +/- 2.6 to 6.8 +/- 1.4 ms/mmHg), the SD of the mean R-R intervals (from 176 +/- 17 to 61 +/- 7 ms), the mean R-R interval (from 742 +/- 32 to 527 +/- 29 ms), and the coefficient of variance [from 235 +/- 15 to 114 +/- 1 (x 1,000)]. The heart rate response to graded submaximal exercise after pertussis toxin was higher (P < 0.001) at each exercise level. In in vitro assay, cardiac tissue samples from pertussis toxin-treated dogs incorporated 10-fold less ADP ribose than what has been described previously. These data prove that the in vivo action of pertussis toxin on cardiac inhibitory G proteins has direct consequences on end-organ cardiac responses to vagal activity. This study quantifies the physiological consequences of pertussis toxin-induced impairment of inhibitory G proteins in conscious animals.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Frequência Cardíaca/fisiologia , Toxina Pertussis , Nervo Vago/fisiologia , Fatores de Virulência de Bordetella/farmacologia , Animais , Cães , Masculino , Esforço Físico , Pressorreceptores/fisiologia , Reflexo/fisiologiaRESUMO
Okadaic acid exerts a positive inotropic effect in cardiac preparations. We studied whether the positive inotropic effect of okadaic acid in cardiac preparations could be due to phosphatase inhibition and whether this inhibition affects the phosphorylation of cardiac proteins. In papillary muscles from guinea pigs, 30 microM okadaic acid increased force of contraction to 175% of predrug value. In isolated guinea pig ventricular cardiomyocytes, okadaic acid augmented single Ca(2+)-channel currents by enhancing channel availability. In homogenates from ventricles, 1 microM okadaic acid completely inhibited phosphorylase a phosphatase activity. In isolated 32P-labeled ventricular cardiomyocytes, 30 microM okadaic acid increased phosphorylation of phospholamban (PLB) and troponin inhibitor (TnI) to 325 and 284% of control, respectively. Furthermore, 30 microM okadaic acid increased phosphorylation of a hitherto unknown 23-kDa protein to 352% of control. It is concluded that the effects of okadaic acid could be mediated by increasing the phosphorylation state of several proteins including PLB, a 23-kDa protein, and TnI.
Assuntos
Éteres Cíclicos/farmacologia , Miocárdio/enzimologia , Fosfoproteínas Fosfatases/fisiologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Separação Celular , Cobaias , Técnicas In Vitro , Isoproterenol/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Ácido Okadáico , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
Langendorff-perfused guinea pig ventricles were used to examine the effects of the adenosine agonists, (-)-N-6-phenylisopropyl-adenosine (PIA) and 5'-N-ethylcarboxamidoadenosine and the muscarinic cholinergic agonist, acetylcholine, on the rate of tension development, protein phosphatase inhibitor-1 (PPI-1) activity, and cyclic AMP-dependent protein kinase (PKA) activity ratio. Isoproterenol (10 nM) and forskolin (1 microM) stimulated rate of tension development, PKA activity ratio and PPI-1 activity each approximately 2-fold. Acetylcholine (1 microM) by itself was not effective, but when administered with isoproterenol for forskolin reduced the rate of tension development and PPI-1 activity without decreasing PKA activity ratio. Similarly, both PIA and 5'-N-ethylcarboxamidoadenosine alone were ineffective, but when simultaneously applied with isoproterenol attenuated the isoproterenol-stimulated rate of tension development and PPI-1 activity. PIA reduced PKA activity ratio, whereas 5'-N-ethylcarboxyamidoadenosine failed to do so. However, the effect of PIA on PKA activity ratio was smaller than those seen on rate of tension development and PPI-1 activity. Hence, the present data do not support a cyclic AMP-dependent regulation of PPI-1 activity by adenosine and muscarinic agonists. It is tempting to speculate that adenosine and muscarinic agonists reduce PPI-1 activity by a cyclic AMP-independent mechanism.
Assuntos
Proteínas de Transporte , Inibidores Enzimáticos/metabolismo , Coração/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Miocárdio/enzimologia , Proteínas/metabolismo , Receptores Muscarínicos/fisiologia , Receptores Purinérgicos/fisiologia , Acetilcolina/farmacologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida) , Animais , Colforsina/farmacologia , Depressão Química , Ativação Enzimática , Feminino , Cobaias , Coração/fisiologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/enzimologia , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Contração Miocárdica/efeitos dos fármacos , Miocárdio/ultraestrutura , Fenilisopropiladenosina/farmacologia , Fosfoproteínas Fosfatases/efeitos dos fármacos , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Proteínas/farmacologia , Receptores Muscarínicos/efeitos dos fármacos , Receptores Purinérgicos/efeitos dos fármacos , Estimulação Química , Função Ventricular , Xantinas/farmacologiaRESUMO
Mechanisms responsible for "denervation supersensitivity" in regionally denervated canine hearts were examined by measuring beta-adrenergic receptor density and affinity and the density of the alpha-subunit of the stimulatory G protein (Gs alpha). Sympathetic denervation was produced by applying an epicardial strip of phenol midway between the left ventricular (LV) base and apex. Six to eight days after denervation, dogs were anesthetized and then underwent functional studies (n = 4) or hearts were excised for biochemical analyses (n = 6). Biochemical studies were also done on 3 nondenervated hearts. Effective refractory periods (ERPs) were measured in innervated (base) and denervated (apex) LV myocardium. During sympathetic stimulation (2 and 4 Hz), the ERP shortened more (P < 0.05) at basal than at apical sites, whereas during norepinephrine infusion (0.05 to 0.5 mg.kg-1 x min-1), the ERP shortened more (P < 0.001) at apical than at basal sites. In regionally denervated hearts, however, the density and affinity of beta-adrenergic receptors did not differ significantly (P > 0.2) in nondenervated basal compared with denervated apical myocardium. Quantitative immunoblotting of the Gs alpha demonstrated that the density of the 47- and 52-kDa subunits was also similar (P > 0.6) in basal compared with apical myocardium from regionally denervated hearts. In addition, beta-adrenergic receptor density and affinity and Gs alpha density did not differ significantly (P > 0.5) in basal compared with apical myocardium from nondenervated control hearts.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Denervação , Coração/inervação , Animais , Cães , Estimulação Elétrica , Coração/efeitos dos fármacos , Immunoblotting , Norepinefrina/farmacologia , Fenol , Fenóis/farmacologia , Receptores Adrenérgicos beta/fisiologia , Transdução de Sinais/fisiologia , Sistema Nervoso Simpático/fisiologiaRESUMO
cAMP content and protein phosphorylation were determined in unlabeled and 32P-labeled guinea pig ventricular myocytes. Isoproterenol (10 nM, 37 degrees C, 10 seconds) increased cAMP content (236%) and phospholamban (265%) and troponin I (135%) phosphorylation in ventricular myocytes. When isoproterenol (0-300 nM) and the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA, 1 microM) or the A1- and A2-adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA, 1 microM) were administered simultaneously, both adenosine receptor agonists attenuated phospholamban phosphorylation to approximately the same extent (40%). The EC50 value for isoproterenol to phosphorylate phospholamban was 8 +/- 1 nM (n = 3), which increased to 31 +/- 4 nM (n = 3) in the presence of PIA or NECA. IC50 values for PIA or NECA to decrease the phosphorylation of phospholamban were 30 or 32 nM in 10 nM isoproterenol-stimulated cells and 80 or 85 nM in 30 nM isoproterenol-stimulated cells. Both adenosine receptor agonists failed to inhibit the phosphorylation of troponin I. However, acetylcholine (2 microM) in the presence of 10 nM isoproterenol inhibited phosphorylation of phospholamban as well as troponin I in ventricular cells. These effects were antagonized by 10 microM atropine. The effects of PIA and NECA on phosphorylation were antagonized by the A1-selective adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (1 microM) but not by the A2-selective adenosine receptor antagonist 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4,triazolo(1,5-c)quinazolin -5-imine (1 microM). PIA and NECA did not reduce cAMP levels in isoproterenol-stimulated cells. We conclude that phospholamban phosphorylation was inhibited by A1-adenosine receptor activation and that these effects on phospholamban phosphorylation cannot be explained by a reduction in cAMP levels.
Assuntos
Acetilcolina/farmacologia , Coração/fisiologia , Isoproterenol/antagonistas & inibidores , Receptores Purinérgicos/fisiologia , Acetilcolina/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida) , Animais , Atropina/farmacologia , Proteínas de Ligação ao Cálcio/química , AMP Cíclico/fisiologia , Feminino , Cobaias , Coração/efeitos dos fármacos , Masculino , Miocárdio/citologia , Fenilisopropiladenosina/farmacologia , Fosforilação/efeitos dos fármacos , Receptores Purinérgicos/efeitos dos fármacos , Troponina/metabolismo , Troponina IRESUMO
The synthesis of a novel radioiodinated ryanodine-O10eq-N-acylamino acylate along with biological data are reported. The affinity of the iodinated product, 7, was comparable to ryanodine, 7.97 nM and 6.47 nM, respectively. Conversion of the non-radioactive iodinated ryanodine analog to the [125I] isotope was accomplished by conversion of 7 to the trimethyltin derivative followed by [125I] exchange using chloramine-T in organic solvent. The radioiodinated ryanodine analog, 9, bound to cardiac membrane preparations in a protein dependent and saturable manner indicating that this analog may represent a useful new tool for the study of ryanodine receptors and that modifications about the C-10 hydroxy group of ryanodine may be carried out without loss in biological activity.
Assuntos
Cálcio/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Receptores Colinérgicos/metabolismo , Rianodina/análogos & derivados , Animais , Cães , Radioisótopos do Iodo , Membranas/metabolismo , Rianodina/química , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de RianodinaRESUMO
The role of G proteins in mediating the responses of the heart to circulating catecholamines and to the influences of the autonomic nervous system is of special interest to cardiologists. It is evident that G proteins are essential links in the cascade of biochemical events that ensure when neurotransmitters and hormones interact with receptors on myocardial cells. It is likely [corrected] that dysfunction of G proteins plays a role in cardiovascular pathophysiology. With current methodologies, especially molecular biological and recombinant DNA techniques, and with transgenic animal models that can relate physiological function and specific gene dosage, some cardiovascular diseases may be traced to G protein-related defects.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Coração/fisiologia , Transdução de Sinais/fisiologia , Animais , Doenças Cardiovasculares/metabolismo , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/química , Sistema de Condução Cardíaco/anatomia & histologia , Humanos , Relação Estrutura-AtividadeRESUMO
The positive inotropic effect of the beta-adrenoceptor agonist isoproterenol is accompanied by inhibition of phosphatase type 1 activity in myocardium. Indirect assays suggest that this effect is due to activation of protein phosphatase inhibitor-1, which inhibits phosphatase activity only when phosphorylated. To test this hypothesis directly, electrically stimulated (3 Hz) guinea pig ventricular preparations were perfused according to the Langendorff method with physiological buffers with or without 5 mCi 32P/heart, and then various concentrations of isoproterenol were applied. Contractility was recorded. Hearts were freeze-clamped and cAMP and inhibitor-1 activities were measured. In 32P-labeled hearts a protein at about 26 kd on autoradiograms of 12% sodium dodecyl sulfate gels was detected. Isoproterenol (1 microM) increased rate of tension development to 238% of the predrug value, cAMP concentrations 1.5-fold, and inhibitor-1 activity threefold. Concomitantly, there was an increase in a 32P-labeled band at about 26 kd from 380 to 540 pmol 32P/mg protein. This protein at about 26 kd, after transfer to nitrocellulose, was recognized by an antiserum prepared against rabbit skeletal muscle inhibitor-1. More radioactive protein of about 26 kd could be immunoprecipitated by the antiserum from isoproterenol-treated than from untreated hearts. It is concluded that a protein, probably identical to phosphatase inhibitor-1, is phosphorylated in vivo in the heart in the presence of isoproterenol. Phosphorylation of inhibitor-1 with consequent modification of type 1 phosphatase activity may contribute to the effects of isoproterenol in the heart.
Assuntos
Proteínas de Transporte , Peptídeos e Proteínas de Sinalização Intracelular , Isoproterenol/farmacologia , Miocárdio/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Proteínas/metabolismo , Animais , Western Blotting , AMP Cíclico/fisiologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/metabolismo , Feminino , Cobaias , Masculino , Peso Molecular , Fosforilação , Proteínas Quinases/metabolismo , Proteínas/química , Proteínas/imunologiaRESUMO
Non-insulin-dependent diabetic (NIDD) male Wistar rats develop a cardiomyopathy approximately 9 mo after the onset of the diabetic condition. This cardiomyopathy is characterized by reduced contractility, relaxation, cardiac work, and diastolic compliance. Although the basis for these defects is not completely understood, altered cellular Ca2+ regulation appears to play a major role in their development. In both isolated sarcolemmal membrane and cardiomyocytes, significant diabetes-linked defects in Ca2+ metabolism were observed. A small, but significant, decrease in the rate of sarcolemmal ATP-dependent Ca2+ transport of the diabetic heart was observed. Also evident was a major defect in sarcolemmal Na(+)-Ca2+ exchange as determined by reduced Na(+)-dependent Ca2+ transport into vesicles and Na(+)-dependent Ca2+ efflux from 45Ca(2+)-loaded cardiomyocytes from diabetic rats. In isolated cardiomyocytes, it was observed that the relative fluorescence of fura-2 at 502 nm was higher in cells from NIDD hearts, suggestive of a higher cytosolic free Ca2+. Consistent with diabetes-linked defects in Ca(2+)-transporter activities, the accumulation of Ca2+ after depolarization with KCl was greater in the diabetic. This study demonstrates that diabetes-induced defects in Ca2+ movement by the various Ca2+ transporters lead to abnormal cytosolic Ca2+ regulation by the diabetic cardiomyocytes. This observation supports the notion that abnormal Ca2+ regulation contributes to the development of the NIDD cardiomyopathy.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Transporte/metabolismo , Sobrevivência Celular , Células Cultivadas , Fura-2 , Coração/efeitos dos fármacos , Cinética , Masculino , Miocárdio/citologia , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Valores de Referência , Sarcolema/metabolismo , Sódio/farmacologia , Trocador de Sódio e Cálcio , Espectrometria de FluorescênciaRESUMO
Muscarinic cholinergic agonists such as acetylcholine attenuate phosphorylation of phospholamban induced by agents that activate cAMP-dependent protein kinase. However, cAMP accumulation is variably affected or only slightly reduced; thus, the choline ester might produce effects in addition to inhibition of adenylate cyclase. We hypothesized that acetylcholine might regulate a phosphatase in mammalina myocardium. Exposure of Langendoff-perfused guinea pig ventricles to isoproterenol (10 nM) for 45 s increased phosphatase inhibitor-1 activity 2-fold. Co-administration of acetylcholine (100 nM) antagonized the effect of isoproterenol, and atropine (1 microM) blocked the effect of acetylcholine. Forskolin (1 microM) caused a 3-fold increase in inhibitor-1 activity, and acetylcholine markedly attenuated the effect of forskolin. However, acetylcholine did not lower cAMP levels in the same tissues. Both isoproterenol and forskolin reduced the type 1 phosphatase activity intrinsic to sarcoplasmic reticulum by 25-50%, using [32P]phosphorylase a or 32P-labeled membrane vesicles as a substrate for the phosphatase. Co-administration of acetylcholine markedly attenuated these effects of isoproterenol and forskolin. Acetylcholine alone caused a 50% increase in type 1 phosphatase activity. We concluded that inhibitor-1 and type 1 phosphatase can be regulated in intact cardiac muscle by agents that increase intracellular cAMP and by acetylcholine.