RESUMO
Nerve terminals ("synaptosomes") isolated from rat brain hippocampus were loaded with the fluorescent Ca2+ indicator fura-2 and were subjected to depolarization with an elevated K+ concentration in a stopped-flow spectrophotometer to measure the activity of voltage-gated Ca2+ channels in the presynaptic membrane. Three components of Ca2+ influx were seen, which were tentatively identified as two classes of voltage-dependent Ca2+ channels with different inactivation kinetics (tau of approximately 60 ms and 1 s, respectively) and Na+/Ca2+ exchange working in the "reverse" mode. The activity of both classes of voltage-dependent Ca2+ channels was slightly augmented by the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage-dependent Ca2+ channel activity, but the effect of DHS could be slowed by coaddition of OKA. These results suggest that the activity of presynaptic voltage-dependent Ca2+ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals.
Assuntos
Canais de Cálcio/fisiologia , Hipocampo/fisiologia , Naftalenos , Terminações Pré-Sinápticas/fisiologia , Proteína Quinase C/metabolismo , Animais , Cálcio/metabolismo , Fura-2 , Cinética , Ésteres de Forbol/farmacologia , Fosforilação , Compostos Policíclicos/farmacologia , Potássio/metabolismo , Proteína Quinase C/antagonistas & inibidores , Ratos , Sódio/metabolismo , Espectrometria de Fluorescência , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
1. Changes of cytoplasmic free calcium levels ([Ca2+]i) in isolated rat brain nerve terminals (synaptosomes), previously loaded with the fluorescent intracellular calcium indicator Fura-2, were measured 1-2 ms after depolarization with elevated K+ by stopped-flow fluorescence spectroscopy. 2. In physiological saline (PSS) containing 4 mM-K+, intraterminal Ca2+ was estimated to be in the range 150-250 nM. Depolarization of the nerve terminals with elevated external K+ in the presence of Ca2+ induced a prompt rise in [Ca2+]i, which occurred in two phases. No change in [Ca2+]i was seen when the terminals were depolarized in nominally Ca(2+)-free solutions, and only a small change was seen when the terminals were acutely exposed to Ca2+ in 4 mM-K+. 3. Predepolarization of the nerve terminals with K+ in nominally Ca(2+)-free solutions several seconds before the introduction of Ca2+ greatly decreased the magnitude of the fast phase, whilst leaving the slow phase largely intact. 4. In Na(+)-depleted nerve terminals, the fast phase of K(+)-stimulated Ca2+ uptake was essentially unaltered, but the slow phase of Ca2+ uptake was dramatically reduced. 5. The rapid phase of K(+)-stimulated uptake displayed voltage-dependent inactivation (tau approximately 50 ms at -10 mV), and the rate of inactivation was accelerated with increasing depolarization. In contrast, at constant [K+]o, increasing [Ca2+]o had little or no effect on the rate of inactivation, but did increase the initial rate of Ca2+ uptake. 6. The dihydropyridine calcium channel blockers nifedipine and nitrendipine had little effect on either component of Ca2+ uptake. However, the inorganic Ca2+ channel blockers La3+, Cd2+, and Co2+ were potent blockers of the fast phase of Ca2+ uptake, but blocked the slow phase only at higher concentrations. No consistent effect of the peptide neurotoxin omega-conotoxin was observed on either component of the Ca2+ rise. 7. These studies demonstrate that the dynamics of depolarization-activated intraterminal Ca2+ changes can be studied on a millisecond time scale in isolated nerve terminals. Moreover, our results indicate that two pathways contribute to depolarization-induced [Ca2+]i changes, namely a voltage-activated, inactivating Ca2+ channel, possibly of the N-type, and Na(+)-Ca2+ exchange operating in the 'reverse' mode.
Assuntos
Cálcio/metabolismo , Sinaptossomos/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Células Cultivadas , Eletrofisiologia , Feminino , Fura-2 , Ativação do Canal Iônico/fisiologia , Cinética , Ratos , Sódio/fisiologiaRESUMO
Fluorescent calcium indicators fluo-3, fura-2 and indo-1, and fluorescent magnesium indicators mag-fura-2 (FURAPTRA) and mag-indo-1 were evaluated for the effects of pH on their association and dissociation rates, ion selectivity and thermodynamic properties. Calcium indicator affinities for Ca and Mg were reduced and the discrimination between Ca and Mg decreased in fura-2 and indo-1 at acidic pH. Alterations in apparent dissociation constants were caused primarily by reduced association rates. Magnesium indicators did not show these changes. The enthalphies of the calcium indicators' Ca complex were 1-3 kcal/mole and magnesium indicators' Mg complex were 7-9 kcal/mole. The potential effects of a biexponential dissociation rate of fluo-3 and of Ca interactions with magnesium indicators were examined.
Assuntos
Cálcio/análise , Corantes Fluorescentes , Magnésio/análise , Compostos de Anilina , Benzofuranos , Fura-2 , Concentração de Íons de Hidrogênio , Indóis , Oxazóis , Espectrometria de Fluorescência/métodos , Termodinâmica , XantenosRESUMO
1. Efflux of 86Rb from synaptosomes prepared from rat forebrain was used to assess voltage-gated changes in K+ permeability in mammalian central nerve terminals. 2. Although they are structurally unrelated to phencyclidine (PCP), the sigma-ligands, N-allyl-normetazocine (NANM; SKF 10,047) and cyclazocine, generalize to PCP in behavioral assays, displace [3H]PCP from a high-affinity binding site in brain, and potently block the same voltage-gated K+ channel as PCP itself. 3. The block of the voltage-gated K+ channel in nerve terminals by NANM and cyclazocine was stereoselective and was unaffected by the opioid antagonist naloxone. Moreover, in our experiments the relative activity of the stereoisomers of NANM and cyclazocine compared favourably with their relative activity in behavioural paradigms and binding assays. 4. Dexoxadrol, the D-isomer of dioxodrol, which produces PCP-like behavioural effects and displaces bound [3H]PCP, was a potent blocker of the PCP-sensitive, voltage-gated K+ channel. The corresponding L-isomer, levoxadrol, which produces morphine-like antinociception and sedation, but does not produce PCP-like behaviour nor displace bound [3H]PCP, was a very weak blocker of the voltage-gated K+ channel. 5. Levoxadrol, but not dexoxadrol, activated a separate K+ channel, as manifested by an increase in 86Rb efflux. This effect was blocked by naloxone. 6. We conclude that one of the PCP-sigma-ligand binding sites in the brain may be associated with the voltage-gated, non-inactivating K+ channel we observe in nerve terminals. Our findings are also consistent with the view that some of the behavioural manifestations of PCP intoxication are mediated by block of presynaptic K+ channels.
Assuntos
Dioxolanos/farmacologia , Dioxóis/farmacologia , Alucinógenos/farmacologia , Fenciclidina/farmacologia , Piperidinas/farmacologia , Canais de Potássio/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Animais , Encéfalo/fisiologia , Ciclazocina/farmacologia , Relação Dose-Resposta a Droga , Técnicas In Vitro , Naloxona/farmacologia , Fenazocina/análogos & derivados , Fenazocina/farmacologia , RatosRESUMO
Phencylidine (PCP) is a major drug of abuse in the United States. It produces a toxic confusional psychosis in man. We show here that nanomolar to micromolar concentrations of PCP and behaviorally active congeners selectively block voltage-regulated noninactivating (or very slowly inactivating) presynaptic K channels in the brain. The rank order of potency for blockage of these K channels parallels both the relative ability of these agents to produce characteristic behavioral deficits in rats and their ability to displace [3H]PCP from its high-affinity binding sites in brain. In view of the enhanced voltage-gated Ca influx that would be expected to accompany blockage of presynaptic K channels, this mechanism could explain the excessive neurotransmitter release that is characteristic of PCP intoxication.
Assuntos
Encéfalo/metabolismo , Canais Iônicos/fisiologia , Fenciclidina/farmacologia , Potássio/metabolismo , Animais , Cálcio/farmacologia , Permeabilidade da Membrana Celular , Relação Dose-Resposta a Droga , Canais Iônicos/efeitos dos fármacos , Fenciclidina/administração & dosagem , Fenciclidina/análogos & derivados , Radioisótopos , Ratos , Rubídio/metabolismo , Sinaptossomos/metabolismoRESUMO
86Rb efflux from pinched-off rat brain presynaptic nerve terminals (synaptosomes) was used to measure the K permeability of the terminals. Synaptosomes were pre-loaded with 86Rb and the suspensions were then filtered on glass fibre filters. The terminals trapped on the filters were superfused with 'efflux solutions', and the effluent and filters were then counted. 86Rb efflux into physiological saline (PSS) containing 5 mM-K and 145 mM-Na was about 0.4% of the 86Rb load per second (component 'R'). Increasing extracellular K concentration [( K]o), or adding veratridine and sea anemone toxin, stimulated efflux; presumably by depolarizing the nerve terminals. The K-stimulated 86Rb efflux was a graded function of [K]o. High [K]o evoked at least three components of efflux: a 'fast phase' (T) that apparently inactivated in less than 1 s, a 'slower phase' (S) that was linear for 3-5 s, and a Ca-dependent phase (C). Some, but not all, of the slow phase 86Rb efflux (component S) may be attributable to increased efflux mediated by the 'resting' K permeability mechanism when the driving force is increased by depolarization. K efflux was also studied and was found to be qualitatively similar to 86Rb efflux. 86Rb: 42K permeability ratios were 0.6-0.8 for most components of the efflux. Raising the Mg concentration in the efflux solution shifted the 86Rb efflux versus [K]o curve in the direction of increased [K]o. This shift may be the result of screening of surface charges by Mg. Several agents that block various K channels in other preparations inhibited K-stimulated 86Rb efflux in synaptosomes: tetraethylammonium (TEA), tetrabutylammonium (TBA), and 4-aminopyridine (4-AP). The fast component (T) of high [K]o-stimulated 86Rb efflux was selectively blocked by low concentrations of 4-AP (apparent half-maximal inhibition, KI = 0.1-0.2 mM); it was also blocked by TEA (KI = 0.6 mM) and TBA (KI = 0.8-1.0 mM). Dose-response curves for inhibition of component T by all three agents were monophasic. the slow component (S) of the K-stimulated 86Rb efflux was much less sensitive to all three agents, than was component T; the broad dose-response curves were consistent with the view that two (or more) different K conductances may contribute to component S.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Encéfalo/citologia , Canais Iônicos/efeitos dos fármacos , Potássio/metabolismo , Sinaptossomos/metabolismo , Animais , Venenos de Cnidários/farmacologia , Feminino , Técnicas In Vitro , Magnésio/farmacologia , Potenciais da Membrana , Permeabilidade , Radioisótopos de Potássio , Radioisótopos , Ratos , Rubídio , Anêmonas-do-Mar , Fatores de Tempo , Veratridina/farmacologiaRESUMO
86Rb efflux was examined in isolated presynaptic nerve terminals (synaptosomes) from rat brain in a study designed to assess K permeability (PK) changes sensitive to alterations in internal Ca activity. Rb efflux from 86Rb-loaded synaptosomes into nominally Ca-free physiological saline (PSS) containing 5 mM-K was about 0.3-0.4%/s. Raising extracellular K concentration [( K]o), to depolarize the synaptosomes, stimulated the 86Rb efflux. Addition of Ca to the 5 mM-K PSS had no effect, but Ca did further stimulate 86Rb efflux into K-rich solutions. The effect of Ca was graded, with apparent half-maximal activation, KA approximately equal to 0.5 mM-Ca. These data fit the view that, during depolarization, Ca enters the terminals through voltage-regulated Ca channels, and that the rise in intracellular Ca concentration opens certain (Ca-activated) K channels. The Ca-dependent stimulation of 86Rb efflux was greatest during the initial seconds of incubation (component CT), and then declined to a much lower rate (component CS). Much of this change in rate could be attributed to inactivation of voltage-regulated Ca channels and reduced entry of Ca. The Ca-dependent increase in 86Rb efflux was completely inhibited by 100 microM-La. In the presence of Ca, but not in its absence, the Ca ionophore A23187 stimulated 86Rb efflux both in 5 and 100 mM-K PSS. The effect in 100 mM-K was quantitatively greater, perhaps because of the increased outward driving force on Rb in depolarized synaptosomes. When synaptosomes were suspended in media containing the voltage-sensitive fluorescent dye, DiS-C3-(5) (1,1'-dipentyl-2,2'-thiocarbocyanine), the addition of Ca+ A23187 decreased the fluorescence intensity (= synaptosome hyperpolarization) when the media contained 5 mM-K but not 100 mM-K. This implies that in the presence of Ca + A23187, PK was increased, and the membrane potential moved closer to the K equilibrium potential, EK. Quinine sulphate, a blocker of Ca-activated K channels, reduced the Ca-stimulated 86Rb efflux with high affinity (apparent half-maximal inhibition, KI approximately equal to 1 microM). Tetraethylammonium chloride, another agent known to block Ca-activated K channels, was also a relatively potent inhibitor of Ca-stimulated 86Rb efflux (KI approximately equal to 0.2 mM). The K-channel blocker, 4-aminopyridine, partially inhibited Ca-stimulated 86Rb efflux at concentrations below 0.5 mM, but stimulated this efflux at concentrations greater than or equal to 1 mM.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Encéfalo/citologia , Cálcio/farmacologia , Canais Iônicos/efeitos dos fármacos , Potássio/metabolismo , Sinaptossomos/metabolismo , 4-Aminopiridina , Aminopiridinas/farmacologia , Animais , Calcimicina/farmacologia , Feminino , Técnicas In Vitro , Lantânio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potássio/farmacologia , Quinina/farmacologia , Radioisótopos , Ratos , RubídioRESUMO
Experiments were carried out to test the hypothesis that membrane depolarization stimulates Ca2+ uptake by vesicles in a sarcolemma preparation. Vesicles from a highly enriched sarcolemma preparation, previously loaded with 150 mM K+, developed a membrane potential when placed in a medium with 2.5 mM K+ as confirmed by changes in fluorescent intensity of the voltage-sensitive dye, 3,3'-dipropyl-2,2'-thiadicarbocyanine. Inclusion of valinomycin in the assay increased the magnitude of the potential, and elevation of extra-vesicular K+, after development of the potential, caused depolarization. Ca2+ uptake by the vesicles was examined under three conditions: (a) nonpolarized state of the vesicles with 150 mM K+ on both sides of the membrane; (b) polarized state of the vesicles with inside negative due to high intravesicular K+ (150 mM) and low extravesicular K+ (2.5 mM); and (c) after a transition from a polarized to a relatively depolarized state by changing extravesicular K+ from 2.5 to about 106 mM. Ca2+ uptake was moderately affected by polarization of the vesicles after 10 min of reaction but was markedly and rapidly enhanced by depolarization of the vesicles. Extravesicular Na+ appeared to be required for the depolarization-induced Ca2+ uptake. La3+, Mn2+, and high concentrations of verapamil and tetrodotoxin inhibited the process. It was concluded that the effect of depolarization on Ca2+ uptake may reflect the process that serves as the trigger for myocardial contraction.
Assuntos
Cálcio/metabolismo , Miocárdio/metabolismo , Sarcolema/fisiologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Cães , Ácido Egtázico/farmacologia , Ventrículos do Coração/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Sódio/farmacologia , Valinomicina/farmacologiaRESUMO
Calcium uptake by vesicles in a highly enriched sarcolemma preparation from canine ventricle was found to be markedly stimulated by intravesicular calcium. Stimulation of calcium uptake appeared to be a saturable function of intravesicular calcium. Calcium efflux from the vesicles was stimulated by calcium in the reaction medium. Calcium uptake, supported by intravesicular calcium, and calcium efflux, stimulated by extravesicular calcium, were found to correspond on a one-to-one basis. Only small changes in net uptake or efflux were observed to occur in response to chemical gradients of calcium across the membrane. It was concluded, therefore, that under certain conditions, the major means for calcium movement across vesicles in the preparation is via a one-to-one exchange of calcium. Sodium was found to stimulate calcium uptake when present in the intravesicular space and to stimulate calcium efflux when present in the extravesicular space, but the effects of calcium plus sodium were not additive with respect to stimulating either calcium uptake or efflux. The effects of unlabeled calcium, strontium, barium, and magnesium on calcium uptake stimulated by intravesicular calcium and by intravesicular sodium were similar though not identical. The temperature dependence for calcium-stimulated and sodium-stimulated calcium movements was characterized by Q10 values of 1.27 and 2.06, respectively. Previous work has associated the sodium-calcium exchange reaction with the sarcolemma. It is argued that the present study, in turn, provides evidence that the calcium-calcium exchange reaction is also associated with the sarcolemma. In addition, the results of the study are consistent with the hypothesis that one membrane system can promote the exchange of either calcium for calcium or calcium for sodium.
Assuntos
Cálcio/metabolismo , Miocárdio/metabolismo , Sarcolema/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Cátions Bivalentes , Cães , Ácido Egtázico/farmacologia , Ventrículos do Coração/metabolismo , Cinética , Organoides/metabolismo , Potássio/farmacologiaRESUMO
A highly enriched sarcolemma preparation was isolated by differential centrifugation of a canine ventricular homogenate followed by centrifugation of a membrane fraction layered over 22% (w/v) sucrose. Ouabain binding, ouabain-sensitive potassium phosphatase activity and 5'-nucleotidase activity were enriched 19--27 fold over the homogenate whereas Ca2+-ATPase and succinate dehydrogenase activities were 0.75 and 0.36, respectively, of that for the homogenate. The isolation procedure was relatively rapid and yielded about 2.0 mg protein/100 g of ventricular muscle. The highest salt concentration used in the procedure was 0.6 M KCl and no detergents were employed. Initial characterization studies suggested that the sarcolemma-enriched fraction consists predominantly if not totally of freely permeable membrane vesicles and that the sarcolemma does not manifest a Ca2+-ATPase activity, at least within the limits of the assay procedures employed. This preparation was concluded to be about 1.5- to 4-fold more highly enriched with sarcolemmal markers than preparations obtained by previously published procedures. Accordingly, the preparation provides an improved basis for the probe of calcium movements that occur across the sarcolemma in association with the excitation-contraction-relaxation sequence of the mammalian myocardial cell.