RESUMO
Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100 microM) and proton pump activity (I50 approximately equal to 15 microM, complete at 100 microM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg2+-ATPase activity, but not proton pumping, was partially restored by N,N'-dicyclohexylcarbodiimide, indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H+-ATPase.
Assuntos
Trifosfato de Adenosina/metabolismo , Medula Suprarrenal/ultraestrutura , Grânulos Cromafim/enzimologia , Sistema Cromafim/enzimologia , Dietilestilbestrol/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Animais , ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , Bovinos , Dienestrol/farmacologia , Hexestrol/farmacologia , Hidrólise , SolubilidadeRESUMO
CaATP is shown to function as a substrate for the proton translocating ATPase of chromaffin granule ghosts at concentrations which are comparable to that of MgATP. Using the initial rate of the proton pump activity as the measure (delta pH/delta t), an apparent Km-value of 139 +/- 8 microM was estimated for CaATP and 59 +/- 3 microM for MgATP. The maximal rate was markedly higher with MgATP than with CaATP, partly due to an inhibition of the hydrolytic activity at the higher concentrations of CaATP. The proton pump activity with CaATP was inhibited by N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide at concentrations similar to that found for MgATP. No inhibition was observed with sodium vanadate in the concentration range 0-15 microM. Calmodulin and trifluoperazine had no effect on the overall ATPase activity with CaATP. These findings establish this activity as an intrinsic property of the chromaffin granules, i.e., linked to the H+-ATPase. No evidence was obtained for the presence of a Ca2+-translocating ATPase [Ca2+ + Mg2+)-ATPase) in the chromaffin granules.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Grânulos Cromafim/enzimologia , Sistema Cromafim/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+) , Cálcio/metabolismo , Cálcio/farmacologia , Calmodulina/farmacologia , Bovinos , Grânulos Cromafim/efeitos dos fármacos , Dicicloexilcarbodi-Imida/farmacologia , Etilmaleimida/farmacologia , Vanadatos , Vanádio/farmacologiaRESUMO
The thiol reagent N-ethylmaleimide (NEM) completely inhibits the proton pump activity of the H+-ATPase in chromaffin granule 'ghosts' at concentrations which only partly (approximately 20%) inhibit the Mg2+-dependent ATP hydrolysis. Half-maximal inhibition was obtained at approximately 13 microM NEM as compared to 18 microM for the classical proton channel inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), and the apparent stoichiometry of the inhibitors at complete inhibition was NEM : DCCD congruent to 1 : 2. HIgh concentrations of NEM (greater than 100 microM) induce a dissipation of the transmembrane potential generated by MgATP. These findings establish NEM as a valuable proton channel inhibitor in chromaffin granules and explain the rather complex effect of NEM previously reported for catecholamine accumulation in this organelle.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/farmacologia , Grânulos Cromafim/enzimologia , Sistema Cromafim/enzimologia , Etilmaleimida/farmacologia , Glândulas Suprarrenais/enzimologia , Animais , Bovinos , Grânulos Cromafim/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , ATPases Translocadoras de PrótonsRESUMO
A subcellular fraction, highly enriched in uncoated vesicles (UCV) with high H+-ATPase (EC 3.6.1.34) activity, was isolated from the crude microsomal fraction of rat liver homogenates by discontinuous sucrose gradient centrifugation. The UCV fraction, recovered at the interface of sucrose density 1.08 and 1.10 g/ml, was shown morphologically to be a mixture of small, smooth-surfaced univesicular and a few multivesicular structures. A permeable anion (e.g. chloride) was required for internal acidification, indicating an electroneutral proton pump. Specific inhibitors of anion transport (pyridoxal 5'-phosphate and 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid) totally inhibit proton translocation. The proton pump activity was insensitive to oligomycin, but was completely inhibited by about 5 microM of the tridentate bathophenanthroline chelate of Fe(II). The activity was also inhibited 100% by low concentrations of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the proton conduction inhibitor N,N'-dicyclohexylcarbodiimide and the ionophore monensin. The UCV fraction contained 2 proteins of Mr 50000 (major) and 54000 (minor) which were phosphorylated by an endogenous cyclic nucleotide- and Ca2+-independent protein kinase.
Assuntos
Microssomos Hepáticos/metabolismo , Fosfoproteínas/isolamento & purificação , ATPases Translocadoras de Prótons/isolamento & purificação , Frações Subcelulares/metabolismo , Adenosina Trifosfatases/isolamento & purificação , Animais , ATPase de Ca(2+) e Mg(2+) , Centrifugação com Gradiente de Concentração , Eletroquímica , Endocitose , Técnicas In Vitro , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/ultraestrutura , Prótons , Ratos , Ratos EndogâmicosRESUMO
We have compared the cardioprotective agents prenylamine and glyceryl trinitrate (GTN) with respect to their effects on the bioenergetics of catecholamine storage vesicles. Chromaffin granule ghosts, which have a well preserved ability to actively transport and store catecholamines, were used as a model for adrenergic synaptic vesicles due to their functional similarity. Prenylamine, which partially and reversibly deplete the endogenous stores of noradrenaline in adrenergic nerves and ganglia, was found to inhibit the generation of the transmembrane proton electrochemical gradient driven by a H(+)-ATPase, mainly by acting as an uncoupler of this ATPase. The inhibition of the energy dependent dopamine uptake (and noradrenaline biosynthesis) by prenylamine could be accounted for by its effect on the bioenergetics of the storage vesicles. The organic nitrates glyceryl trinitrate and isosorbide dinitrate also partly inhibited the catecholamine uptake in parallel with their effects on the proton electrochemical gradient. It is concluded that GTN is a weak catecholamine depletor. Experiments with 3-morpholinosydnonimin-hydrochloride, a source of nitric oxide (NO), opens up the possibility that the mechanism of inhibition of the bioenergetics of chromaffin granule ghosts by GTN is mediated by NO.
Assuntos
Catecolaminas/metabolismo , Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , Metabolismo Energético/efeitos dos fármacos , Nitroglicerina/farmacologia , Prenilamina/farmacologia , Animais , Bovinos , Dopamina/metabolismo , Técnicas In Vitro , Dinitrato de Isossorbida/análogos & derivados , Dinitrato de Isossorbida/farmacologia , ATPases Translocadoras de Prótons/análiseRESUMO
A number of commonly used calcium channel blockers have been compared with respect to their effects on the bioenergetics of catecholamine storage vesicles. Chromaffin granule ghosts with a well-preserved ability to actively transport and store catecholamines, were used as a model for adrenergic synaptic vesicles due to their functional similarity. Nicardipine, verapamil, terodiline and diltiazem were found to have effects comparable to that of prenylamine (Grønberg, M., O. Terland, E.S. Husebye and T. Flatmark, 1990. Biochem. Pharmacol. 40, 351) by inhibiting the generation of a transmembrane proton electrochemical gradient driven by the vesicular H(+)-ATPase, mainly by loose-coupling/uncoupling of this ATPase. Amlodipine inhibited the internal acidification of the vesicles in a tyramine-like manner and increased the steady-state membrane potential (positive inside) generated by the MgATP-dependent proton translocation. Nifedipine and felodipine also inhibited the efficiency of the proton pump, but their mechanisms of action require further investigation. The concentrations giving 50% inhibition of the H(+)-ATPase-dependent generation of a pH-gradient were found to be: 12 microM felodipine, 16 microM nicardipine, 25 microM terodiline, 50 microM nifedipine, 60 microM verapamil, 65 microM amlodipine and 150 microM diltiazem. The effects of the calcium channel blockers on the bioenergetics of chromaffin granules explain the release of catecholamines from sympathetic nerves and ganglia in vitro by the calcium channel blockers.
Assuntos
Medula Suprarrenal/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Catecolaminas/metabolismo , Metabolismo Energético/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , Medula Suprarrenal/efeitos dos fármacos , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Bovinos , Sistema Cromafim/efeitos dos fármacos , Sistema Cromafim/enzimologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Prenilamina/farmacologiaRESUMO
The Mg2+-ATPase activities of bovine adrenal chromaffin granules were studied in highly purified preparations of granule ghosts and in intact organelles. The overall ATPase activity (150-250 nmol ADP min-1 mg-1) of the granule ghost preparations was inhibited less than 5% by the bathophenanthroline chelate of Fe(II), a potent inhibitor of mitochondrial F1-ATPase. This small inhibition can be accounted for by a very minor contamination with mitochondria or mitochondrial fragments. The overall ATPase activity of native granule ghosts was inhibited about 75% by N-ethylmaleimide, with half-maximal inhibition at about 20 microM. The titration curve was slightly shifted towards higher concentrations as compared to the inhibition curve for the proton pump activity, which was completely inhibited at 25 microM. N,N'-Dicyclohexylcarbodiimide inhibited the overall ATPase activity by 75-80% at 1.1 mumol/mg protein, a concentration that completely abolished the proton pump activity. Low concentrations (10 microM) of vanadate inhibited the overall ATPase activity by about 15% but had no effect on the proton pump activity, which was partly inhibited only at higher vanadate concentrations. Our attempts to assign a function to the vanadate-sensitive and N-ethylmaleimide-insensitive ATPase have so far been unsuccessful. In particular, our assay for ATP diphosphohydrolase activity was negative, although the chromaffin granule ghosts revealed a low Mg2+-ADPase activity (11.8 nmol AMP min-1 mg-1 protein). In intact chromaffin granules the specific Mg2+-ATPase activity (50-70 nmol ADP min-1 mg-1) was stimulated 2-fold by uncouplers, as compared to 1.6-1.7-fold in granule ghosts. The degree of energy coupling was rather independent of the external pH (6.5 less than pH less than 8.0) and temperature (20-45 degrees C). As expected, partial inhibition (about 15%) of the overall ATPase activity by 10 microM vanadate increased the ATPase control ratio. ADP was found to be a potent inhibitor of the proton pump activity with MgATP as the substrate, and the effect can partly be explained by a competitive type of inhibition of the hydrolytic reaction. This effect of ADP explains some of the kinetic data reported for MgATP-dependent (H+-ATPase-dependent) reactions in this organelle, notably the energy-dependent accumulation and storage of catecholamines.
Assuntos
Medula Suprarrenal/citologia , ATPase de Ca(2+) e Mg(2+)/metabolismo , Grânulos Cromafim/enzimologia , Sistema Cromafim/enzimologia , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Medula Suprarrenal/enzimologia , Animais , ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , Bovinos , Dicicloexilcarbodi-Imida/farmacologia , Etilmaleimida/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Fenantrolinas/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Temperatura , Vanadatos , Vanádio/farmacologiaRESUMO
Dopamine (100 microM, 10-30 min) inhibits/inactivates the MgATP-dependent generation of a transmembrane proton electrochemical gradient in chromaffin granule ghosts. The dopamine dependent inhibition was enhanced by adding soluble dopamine beta-monooxygenase (DBM, 0.2 U/ml) and completely prevented by ascorbate (1 mM), dithiothreitol (2 mM) and approximately 80% by the DBM inhibitor fusaric acid (10 microM). This indicates that the inhibition is caused by the dopamine semiquinone free radical generated during DBM-dependent dopamine oxidation. Catalase, superoxide dismutase or both did not prevent the inhibition, and DBM-catalysed dopamine oxidation did not change the basal level of lipid peroxidation, excluding the involvement of reactive oxygen species as being responsible for the inhibition. N-ethylmaleimide-sensitive ATPase activity (i.e. the proton translocating ATPase) in the vesicle membranes was inhibited during dopamine incubation, indicating that the toxic metabolite (dopamine semiquinone) inhibits proton pumping by inhibiting the endogenous vacuolar H(+)-ATPase. As this proton pump represents the driving force for the vesicular uptake and storage of catecholamines, the dopamine dependent inhibition, if taking place in vivo, may inhibit dopamine uptake in storage vesicles in sympathetic neurons, e.g. as observed in the myopathic hamster heart.