RESUMO
Immunocytochemistry reveals that the Na/Ca exchanger (NCX) in neuronal somata and astrocytes is confined to plasma membrane (PM) microdomains that overlie sub-PM (junctional) endoplasmic reticulum (jER). By contrast, the PM Ca(2+) pump (PMCA) is more uniformly distributed in the PM. At presynaptic nerve terminals, the NCX distribution is consistent with that observed in the neuronal somata, but the PMCA is clustered at the active zones. Thus, the PMCA, with high affinity for Ca(2+) (K(d) congruent with 100 nM), may keep active zone Ca(2+) very low and thereby "reprime" the vesicular release mechanism following activity. NCX, with lower affinity for Ca(2+) (K(d) congruent with 1,000 nM), on the other hand, may extrude Ca(2+) that has diffused away from the active zones and been temporarily sequestered in the endoplasmic reticulum. The PL microdomains that contain the NCX also contain Na(+) pump high ouabain affinity alpha2 (astrocytes) or alpha 3 (neurons) subunit isoforms (IC(50) congruent with 5-50 nM ouabain). In contrast, the alpha1 isoform (low ouabain affinity in rodents; IC(50) >10,000 nM), like the PMCA, is more uniformly distributed in these cells. The sub-PM endoplasmic reticulum in neurons (and probably glia and other cell types as well) and the adjacent PM form junctions that resemble cardiac muscle dyads. We suggest that the PM microdomains containing NCX and alpha 2/alpha 3 Na(+) pumps, the underlying jER, and the intervening tiny volume of cytosol (<10(-18) l) form functional units (PLasmERosomes); diffusion of Na(+) and Ca(2+) between these cytosolic compartments and "bulk" cytosol may be markedly restricted. The activity of the Na(+) pumps with alpha 2/alpha 3 subunits may thus regulate NCX activity and jER Ca(2+) content. This view is supported by studies in mice with genetically reduced (by congruent with 50%) alpha 2 Na(+) pumps: evoked Ca(2+) transients were augmented in these cells despite normal cytosolic Na(+) and resting Ca(2+) concentrations ([Na(+)](CYT) and [Ca(2+)](CYT)). We conclude that alpha 2/alpha 3 Na(+) pumps control PLasmERosome (local) [Na(+)](CYT). This, in turn, via NCX, modulates local [Ca(2+)](CYT), jER Ca(2+) storage, Ca(2+) signaling, and cell responses.
Assuntos
Astrócitos/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/metabolismo , Neurônios/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Encéfalo/metabolismo , Isoenzimas/metabolismo , Cinética , Camundongos , Modelos Biológicos , Subunidades Proteicas/metabolismoRESUMO
Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter-lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity.
Assuntos
Inibidores da Captação de Dopamina/toxicidade , Dopamina/análise , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Perfilação da Expressão Gênica , Biblioteca Gênica , Metanfetamina/toxicidade , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ácido 3,4-Di-Hidroxifenilacético/análise , Animais , DNA Complementar/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Indução Enzimática , Feminino , Genes Sintéticos , Óperon Lac , Masculino , Mesencéfalo/citologia , Mesencéfalo/embriologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Ratos , Transcrição Gênica , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Pulmonary vasoconstriction and vascular medial hypertrophy greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. A rise in cytosolic free Ca(2+) ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (E(m)) regulates [Ca(2+)](cyt) by governing Ca(2+) influx through voltage-dependent Ca(2+) channels. Thus intracellular Ca(2+) may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling. In PASMC, activity of voltage-gated K(+) (Kv) channels regulates resting E(m). In this study, we investigated whether changes of Kv currents [I(K(V))], E(m), and [Ca(2+)](cyt) affect cell growth by comparing these parameters in proliferating and growth-arrested PASMC. Serum deprivation induced growth arrest of PASMC, whereas chelation of extracellular Ca(2+) abolished PASMC growth. Resting [Ca(2+)](cyt) was significantly higher, and resting E(m) was more depolarized, in proliferating PASMC than in growth-arrested cells. Consistently, whole cell I(K(V)) was significantly attenuated in PASMC during proliferation. Furthermore, E(m) depolarization significantly increased resting [Ca(2+)](cyt) and augmented agonist-mediated rises in [Ca(2+)](cyt) in the absence of extracellular Ca(2+). These results demonstrate that reduced I(K(V)), depolarized E(m), and elevated [Ca(2+)](cyt) may play a critical role in stimulating PASMC proliferation. Pulmonary vascular medial hypertrophy in patients with pulmonary hypertension may be partly caused by a membrane depolarization-mediated increase in [Ca(2+)](cyt) in PASMC.
Assuntos
Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Artéria Pulmonar/citologia , Artéria Pulmonar/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Fenômenos Fisiológicos Sanguíneos , Cálcio/metabolismo , Divisão Celular/fisiologia , Células Cultivadas , Quelantes/farmacologia , Meios de Cultura/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Eletrofisiologia , Espaço Extracelular/metabolismo , Ativação do Canal Iônico/fisiologia , Ionóforos/farmacologia , Potássio/metabolismo , Canais de Potássio/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
A partially purified preparation of the lobster muscle Na(+)/Ca(2+) exchanger was reconstituted with, presumably, random orientation in liposomes. Ca(2+) efflux from (45)Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na(+) (Na(ev))-dependent Ca(2+) efflux depended directly upon the extravesicular Ca(2+) concentration ([Ca(2+)](ev)) with a half-maximal activation at [Ca(2+)](ev) = 0.6 microm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca(2+), as in most other species. In contrast, at low [Na(+)](ev), the Ca(ev)-binding site (i.e., on the cytoplasmic surface) for Ca(2+) transported via Ca(2+)/Ca(2+) exchange was half-maximally activated by about 7.5 microm Ca(2+). Mild proteolysis of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin also upregulated the Na(ev)-dependent Ca(2+) efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca(2+). Proteolytic digestion in the presence of 1.9 microm free ev Ca(2+), however, induced only a 1. 6-fold augmentation of Ca(2+) efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca(2+) also inhibited proteolytic degradation of the Na(+)/Ca(2+) exchanger measured by immunoblotting. These data suggest that Ca(2+), bound to a high affinity binding site, protects against the activation of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin. Additionally, we observed a 6-fold increase in the Na(+)/Ca(2+) exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids.
Assuntos
Músculo Esquelético/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Radioisótopos de Cálcio/metabolismo , Quimotripsina/metabolismo , Citoplasma/metabolismo , Nephropidae , ProteolipídeosRESUMO
The plasma membrane ATP-driven Ca2+ pump (PMCA) and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion at presynaptic nerve terminals, but little is know about the location of these transporters relative to the major sites of Ca2+ influx, the transmitter release sites. We used immunocytochemistry to identify these transport proteins in a calyx-type presynaptic nerve terminal from the ciliary ganglion of the chick. The PMCA clusters were localized to the transmitter release sites, as identified by staining for the secretory vesicle-specific protein synaptotagmin I. This colocalization was not due to the presence of the pump on the secretory vesicle itself because membrane fractionation of chick brain synaptosomes demonstrated comigration of the pump with surface membrane and not vesicle markers. In contrast, the NCX did not colocalize with synaptotagmin but tended to be located at nonsynaptic regions of the terminal. The PMCA location, near the transmitter release sites, suggests that it plays a role in priming the release site by maintaining a low free Ca2+ level, facilitating the dissociation of the ion from its binding sites. The PMCA may also replenish external Ca2+ in the synaptic cleft following periods of synaptic activity. In contrast, the NCX location suggests a role in the rapid emptying of cytoplasmic Ca2+ uptake organelles which serve as the main line of defence against high free Ca2+.
Assuntos
Proteínas de Ligação ao Cálcio , ATPases Transportadoras de Cálcio/metabolismo , Terminações Pré-Sinápticas/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Western Blotting , Encéfalo/citologia , Embrião de Galinha , Gânglios Parassimpáticos/citologia , Gânglios Parassimpáticos/metabolismo , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Terminações Nervosas/enzimologia , Terminações Nervosas/metabolismo , Terminações Nervosas/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/enzimologia , Terminações Pré-Sinápticas/ultraestrutura , Sinaptossomos/metabolismo , Sinaptotagmina I , SinaptotagminasAssuntos
Coração/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Trocador de Sódio e Cálcio/biossíntese , Animais , Artérias/citologia , Cálcio/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Desenho de Fármacos , Expressão Gênica/efeitos dos fármacos , Meia-Vida , Músculo Liso Vascular/citologia , Miocárdio/citologia , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/genéticaRESUMO
BACKGROUND: Primary pulmonary hypertension (PPH) is a rare disease of unknown cause. Although PPH and secondary pulmonary hypertension (SPH) share many clinical and pathological characteristics, their origins may be disparate. In pulmonary artery smooth muscle cells (PASMCs), the activity of voltage-gated K+ (KV) channels governs membrane potential (Em) and regulates cytosolic free Ca2+ concentration ([Ca2+]cyt). A rise in [Ca2+]cyt is a trigger of vasoconstriction and a stimulus of smooth muscle proliferation. METHODS AND RESULTS: Fluorescence microscopy and patch clamp techniques were used to measure [Ca2+]cyt, Em, and KV currents in PASMCs. Mean pulmonary arterial pressures were comparable (46+/-4 and 53+/-4 mm Hg; P=0.30) in SPH and PPH patients. However, PPH-PASMCs had a higher resting [Ca2+]cyt than cells from patients with SPH and nonpulmonary hypertension disease. Consistently, PPH-PASMCs had a more depolarized Em than SPH-PASMCs. Furthermore, KV currents were significantly diminished in PPH-PASMCs. Because of the dysfunctional KV channels, the response of [Ca2+]cyt to the KV channel blocker 4-aminopyridine was significantly attenuated in PPH-PASMCs, whereas the response to 60 mmol/L K+ was comparable to that in SPH-PASMCs. CONCLUSIONS: These results indicate that KV channel function in PPH-PASMCs is inhibited compared with SPH-PASMCs. The resulting membrane depolarization and increase in [Ca2+]cyt lead to pulmonary vasoconstriction and PASMC proliferation. Our data suggest that defects in PASMC KV channels in PPH patients may be a unique mechanism involved in initiating and maintaining pulmonary vasoconstriction and appear to play a role in the pathogenesis of PPH.
Assuntos
Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Canais de Potássio/deficiência , Potássio/metabolismo , Artéria Pulmonar/metabolismo , Adulto , Idoso , Cálcio/metabolismo , Células Cultivadas , Feminino , Hemodinâmica , Humanos , Transporte de Íons , Masculino , Potenciais da Membrana , Microscopia de Fluorescência , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Vasoconstrição/fisiologiaAssuntos
Depressores do Apetite/farmacologia , Fenfluramina/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Expressão Gênica , Humanos , Técnicas In Vitro , Músculo Liso Vascular/citologia , Canais de Potássio/biossíntese , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , RNA Mensageiro/genéticaRESUMO
Arterial smooth muscle (ASM) contraction is triggered by agonist-evoked Ca2+ mobilization from sarcoplasmic reticulum (SR). The amount of Ca2+ released, and thus, the magnitude of the contractions, depends directly on SR Ca2+ content. Na+ pump inhibition by cardiotonic steroids (CTS) indirectly increases the Ca2+ content of the SR and, thus, contractility. This sequence of events does not, however, account for the multiple Na+ pump alpha subunit isoforms with different affinities for Na+ and for CTS, nor does it explain the cardiotonic and vasotonic effects of low doses of CTS that do not elevate cytosolic Na+ or Ca2+. We show that the Na+ pump high ouabain affinity (alpha3) isoform and the plasmalemmal (PM) Na/Ca exchanger are confined to PM domains that overlie junctional SR in ASM, while low ouabain affinity alpha1 and the PM Ca2+ pump are uniformly distributed in the PM. Thus, low doses of CTS, including an endogenous ouabain-like compound, influence cytosolic Na+ and (indirectly) Ca2+ concentrations only in the cytoplasmic clefts between the PM and junctional SR (a functional unit we call the "plasmerosome"). In turn, this modulates the Ca2+ content of the junctional SR and cell responsiveness.
Assuntos
Glicosídeos Cardíacos/farmacologia , Inibidores Enzimáticos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Artérias/citologia , Artérias/efeitos dos fármacos , Artérias/metabolismo , Cálcio/metabolismo , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
K(+)-channel activity-mediated alteration of the membrane potential and cytoplasmic free Ca2+ concentration ([Ca2+]cyt) is a pivotal mechanism in controlling pulmonary vasomotor tone. By using combined approaches of patch clamp, imaging fluorescent microscopy, and molecular biology, we examined the electrophysiological properties of K+ channels and the role of different K+ currents in regulating [Ca2+]cyt and explored the molecular identification of voltage-gated K+ (KV)- and Ca(2+)-activated K+ (KCa)-channel genes expressed in pulmonary arterial smooth muscle cells (PASMC). Two kinetically distinct KV currents [IK(V)], a rapidly inactivating (A-type) and a noninactivating delayed rectifier, as well as a slowly activated KCa current [IK(Ca)] were identified. IK(V) was reversibly inhibited by 4-aminopyridine (5 mM), whereas IK(Ca) was significantly inhibited by charybdotoxin (10-20 nM). K+ channels are composed of pore-forming alpha-subunits and auxiliary beta-subunits. Five KV-channel alpha-subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel alpha-subunit gene from the Shab subfamily (KV2.1), a KV-channel modulatory alpha-subunit (KV9.3), and a KCa-channel alpha-subunit gene (rSlo), as well as three KV-channel beta-subunit genes (KV beta 1.1, KV beta 2, and KV beta 3) are expressed in PASMC. The data suggest that 1) native K+ channels in PASMC are encoded by multiple genes; 2) the delayed rectifier IK(V) may be generated by the KV1.1, KV1.2, KV1.5, KV1.6, KV2.1, and/or KV2.1/KV9.3 channels; 3) the A-type IK(V) may be generated by the KV1.4 channel and/or the delayed rectifier KV channels (KV1 subfamily) associated with beta-subunits; and 4) the IK(Ca) may be generated by the rSlo gene product. The function of the KV channels plays an important role in the regulation of membrane potential and [Ca2+]cyt in PASMC.
Assuntos
Ativação do Canal Iônico/fisiologia , Músculo Liso Vascular/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Artéria Pulmonar/metabolismo , Animais , Cálcio/metabolismo , Cálcio/fisiologia , Células Cultivadas , Citoplasma/metabolismo , Canais de Potássio de Retificação Tardia , Eletrofisiologia , Immunoblotting , Músculo Liso Vascular/citologia , Concentração Osmolar , Canais de Potássio/genética , Artéria Pulmonar/citologia , Ratos , Canais de Potássio Shab , Transcrição Gênica/fisiologiaAssuntos
Hipertensão Pulmonar/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Canal de Potássio Kv1.5 , Canais de Potássio/metabolismo , RNA Mensageiro/metabolismo , Transcrição GênicaAssuntos
Astrócitos/enzimologia , Membrana Celular/enzimologia , Músculo Liso Vascular/enzimologia , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Animais Recém-Nascidos , Aorta/citologia , Aorta/enzimologia , Astrócitos/citologia , Encéfalo/enzimologia , Células Cultivadas , Isoenzimas/química , Isoenzimas/metabolismo , Substâncias Macromoleculares , Artérias Mesentéricas/citologia , Artérias Mesentéricas/enzimologia , RatosRESUMO
Activity of voltage-gated K+ channels (KV) in pulmonary arterial smooth muscle cells (PASMC) is pivotal in controlling membrane potential, cytoplasmic free Ca2+ concentration ([Ca2+]cyt, and pulmonary vasomotor tone. Acute hypoxia selectively inhibits KV channels, depolarizes PASMC, raises [Ca2+]cyt, and causes pulmonary vasoconstriction and vascular remodeling. Prolonged hypoxia (24-60 h) decreased significantly the mRNA levels of KV channel alpha subunits, KV1.2 and KV1.5. Consistently, the protein levels of KV1.2 and KV1.5 were also decreased significantly by hypoxia (48-72 h). Nevertheless, hypoxia affected negligibly the mRNA levels of KV channel beta subunits (KVbeta1, KVbeta2, and KVbeta3). The native K+ channels are composed of pore-forming alpha and auxiliary beta subunits. Assembly of KV beta subunits with alpha subunits confers rapid inactivation on the slowly or non-inactivating delayed rectifier KV channels. KV beta subunits also function as an open-channel blocker of KV channels. Thus, the diminished transcription and expression of KV alpha subunits may reduce the number of KV channels and decrease KC currents. Unchanged transcription of KV beta subunits may increase the fraction of the KV channel alpha subunits that are associated with beta subunits and further reduce the total KV currents. These data demonstrate a novel mechanism by which chronic hypoxia may cause pulmonary vasoconstriction and hypertension.
Assuntos
Hipóxia/genética , Músculo Liso Vascular/fisiologia , Canais de Potássio/genética , Animais , Células Cultivadas , Regulação para Baixo , Expressão Gênica , Canais de Potássio/metabolismo , Artéria Pulmonar/fisiologia , RNA Mensageiro/genética , Ratos , Transcrição GênicaRESUMO
The objective of this study was to determine the effect of angiotensin I (Ang I) treatment in vivo on two major Ca-transport systems-the L-type voltage dependent calcium channel (L-VDCC) and the Na/Ca exchanger in rat heart. For our experiments we used four groups of rats, treated differently with saline, Ang I, the ACE inhibitor enalapril and/or combination of both for 6 days, every 24 h. We observed an increase in the activity, and also in mRNA expression of the Na/Ca exchanger, after repeated administration of Ang I in vivo. The maximal binding capacity of Ca-antagonist PN 200-110, which binds to the alpha 1 subunit of the L-VDCC was elevated from 0.8-1.85 pg/mg protein. mRNA expression of the voltage-dependent calcium channels of L-type system was also upregulated by Ang I administration, but not when enalapril was applied simultaneously with Ang I. These results demonstrate that in vivo application of the Ang I significantly modulates not only the activity, but also expression of the Na/Ca exchanger and the L-VDCC in rat hearts through angiotensin II (Ang II). Since in the in vitro experiments on the isolated cardiomyocytes, Ang II (100 nM) increased the calcium uptake after depolarization, and the AT1 receptor agonist losartan prevented this increase, we assume that this regulation might involve the AT1 receptors.
Assuntos
Angiotensina II/metabolismo , Angiotensina I/farmacologia , Cálcio/metabolismo , Miocárdio/metabolismo , Actinas/metabolismo , Angiotensina I/metabolismo , Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Transporte Biológico , Northern Blotting , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L , Células Cultivadas , Enalapril/farmacologia , Coração/efeitos dos fármacos , Hibridização In Situ , Isradipino/metabolismo , Masculino , Miocárdio/citologia , Ratos , Ratos Wistar , Receptores de Angiotensina/metabolismo , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Three isoforms (alpha1, alpha2, and alpha3) of the catalytic (alpha) subunit of the plasma membrane (PM) Na+ pump have been identified in the tissues of birds and mammals. These isoforms differ in their affinities for ions and for the Na+ pump inhibitor, ouabain. In the rat, alpha1 has an unusually low affinity for ouabain. The PM of most rat cells contains both low (alpha1) and high (alpha2 or alpha3) ouabain affinity isoforms, but precise localization of specific isoforms, and their functional significance, are unknown. We employed high resolution immunocytochemical techniques to localize alpha subunit isoforms in primary cultured rat astrocytes, neurons, and arterial myocytes. Isoform alpha1 was ubiquitously distributed over the surfaces of these cells. In contrast, high ouabain affinity isoforms (alpha2 in astrocytes, alpha3 in neurons and myocytes) were confined to a reticular distribution within the PM that paralleled underlying endoplasmic or sarcoplasmic reticulum. This distribution is identical to that of the PM Na/Ca exchanger. This raises the possibility that alpha1 may regulate bulk cytosolic Na+, whereas alpha2 and alpha3 may regulate Na+ and, indirectly, Ca2+ in a restricted cytosolic space between the PM and reticulum. The high ouabain affinity Na+ pumps may thereby modulate reticulum Ca2+ content and Ca2+ signaling.
Assuntos
Astrócitos/metabolismo , Músculo Liso Vascular/metabolismo , Neurônios/metabolismo , Ouabaína/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Anticorpos/imunologia , Western Blotting , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Músculo Liso Vascular/citologia , Neurônios/citologia , Ouabaína/farmacologia , Ratos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
The effects of chimeric phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted against the Na+/Ca2+ exchanger (NCX) were tested in primary cultured rat mesenteric artery myocytes. In parallel cultures, myocytes proliferated and were morphologically normal in the presence of scrambled nonsense (NS-) or AS-oligos or no oligos (controls). NCX function was examined with digital imaging, using fura 2 to estimate the cytosolic free Ca2+ concentration ([Ca2+]cyt). Resting [Ca2+]cyt was higher (145 +/- 4 nM; P < 0.05) in AS-oligo-treated cells than in controls (125 +/- 5 nM) or NS-oligo-treated cells (131 +/- 4 nM). Lowering external Na+, to promote Ca2+ entry via NCX, increased [Ca2+]cyt transiently in controls and NS-oligo-treated cells but not in AS-oligo-treated cells. Raising the cytosolic free Na+ concentration with ouabain augmented the low-Na(+)-induced rise in [Ca2+]cyt in controls and NS-oligo-treated cells, but AS-oligo-treated cells still did not respond. Nevertheless, serotonin (5-HT) increased [Ca2+]cyt in all three groups. Thus AS-oligos selectively blocked NCX activity but not the 5-HT response. To determine the effect of NCX knockdown on the modulation of stored Ca2+, the 5-HT response was tested immediately after removal of external Ca2+. Ouabain augmented the 5-HT-induced rise in [Ca2+]cyt in control and NS-oligo-treated cells but not AS-oligo-treated cells. This indicates that the NCX can modulate intracellular Ca2+ stores. We conclude that AS-oligos are useful for investigating the physiological role of NCX in vascular smooth muscle.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Artérias Mesentéricas/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Animais , Sequência de Bases , Cálcio/agonistas , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citosol/metabolismo , Membranas Intracelulares/metabolismo , Artérias Mesentéricas/citologia , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/genética , Ratos , Trocador de Sódio e CálcioRESUMO
The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger. Immunoblots of the lobster muscle and mammalian heart exchangers, following SDS-PAGE, indicate that the invertebrate and mammalian exchangers have similar molecular weights: about 120 kDa. The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin vesicles which were loaded with 160 mM NaCl. 45Ca uptake by these proteoliposomes was promoted by replacing 160 mM NaCl in the external medium with 160 mM KCl to produce an outwardly-directed Na+ concentration gradient. When the proteoliposomes were adsorbed onto black lipid membranes (BLM), and DM-Nitrophen-Ca2+ ("caged Ca2+") was added to the KCl medium, photolytically-evoked Ca2+ concentration jumps elicited transient electric currents. These currents corresponded to positive charge exiting from the proteoliposomes, and were consistent with the Na/Ca exchanger-mediated exit of 3 Na+ in exchange for 1 entering Ca2+. The current was dependent upon the Ca2+ concentration jump, the protein integrity, and the outwardly directed Na+ gradient. KCl-loaded proteoliposomes did not produce any current. Low external Na+ concentrations augmented the current, whereas Na+ concentrations > 25 mM reduced the current. The dependence of the current on free Ca2+ was Michaelis-Menten-like, with half-maximal activation (KM(Ca)) at < 10 microM Ca2+. Caged Sr2+ and Ba2+, but not Mg2+, also supported photolysis-evoked outward current, as did Ni2+, but not Mn2+. However, Mg2+ and Mn2+ augmented the Ca-dependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La3+ (added as 200 microM DMN-La3+). The results indicate that the properties of the Na/Ca exchanger can be studied with these electro-physiological methods.
Assuntos
Acetatos/efeitos da radiação , Cálcio/metabolismo , Proteínas de Transporte/fisiologia , Etilenodiaminas/efeitos da radiação , Proteínas Musculares/fisiologia , Nephropidae/metabolismo , Fotólise , Proteolipídeos , Adsorção , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/isolamento & purificação , Cátions Bivalentes/farmacologia , Cinética , Lantânio/farmacologia , Bicamadas Lipídicas , Potenciais da Membrana/efeitos da radiação , Proteínas Musculares/isolamento & purificação , Fosfatidilcolinas , Fosfolipídeos , Cloreto de Potássio/metabolismo , Cloreto de Sódio/metabolismo , Trocador de Sódio e Cálcio , Raios UltravioletaRESUMO
Regulation of the cytosolic free Ca2+ concentration ([Ca2+]cyt) by an Na/Ca exchanger was studied in primary cultured rat cortical astrocytes. [Ca2+]cyt was measured by digital imaging in cells loaded with fura-2. The resting [Ca2+]cyt, approximately 150 nM, was only slightly increased by reducing the extracellular Na+ concentration ([Na+]o) to 6.2 mM, or by treating the cells with ouabain for 15 min (to raise cytosolic Na+). Following treatment with ouabain, however, lowering [Na+]o caused [Ca2+]cyt to rise rapidly to approximately 1300 nM. When Ca2+ sequestration in intracellular stores was blocked by thapsigargin, lowering [Na+]o increased [Ca2+]cyt to approximately 1500 nM in the absence of ouabain. The low-[Na+]o-stimulated rise in [Ca2+]cyt was abolished by removal of external Ca2+, but was not blocked by the Ca2+ channel blocker verapamil, or by caffeine or ryanodine, which deplete an intracellular Ca2+ store responsible for Ca(2+)-induced Ca2+ release. These data suggest that Na+ gradient reduction promotes net Ca2+ gain via Na/Ca exchange. Normally, however, a large rise in [Ca2+]cyt is prevented by sequestration of the entering Ca2+; this buffering of cytosolic Ca2+ can be circumvented by blocking sequestration with thapsigargin, or overwhelmed by enhancing net Ca2+ gain by pretreating the cells with ouabain. The presence of Na/Ca exchanger protein and mRNA in the astrocytes was confirmed by Western and Northern blot analyses, respectively. Immunohistochemistry revealed that exchanger molecules are distributed in a reticular pattern over the astrocyte surface. We suggest that the Na/Ca exchanger plays a role in regulating both [Ca2+]cyt and the intracellular stores of Ca2+ in astrocytes, and may thus contribute to the control of astrocyte responsiveness to neurotransmitters and neurotoxins.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/análise , Córtex Cerebral/metabolismo , Sódio/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/química , Astrócitos/efeitos dos fármacos , Cafeína/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Citosol/metabolismo , DNA Complementar/análise , Transporte de Íons , Microscopia de Fluorescência , Ouabaína/farmacologia , RNA/análise , RNA Mensageiro/análise , Ratos , Rianodina/farmacologia , Terpenos/farmacologia , Tapsigargina , Verapamil/farmacologiaRESUMO
Antibodies raised against dog cardiac Na(+)-Ca2+ exchanger were employed to determine the presence and distribution of the exchanger in arterial smooth muscle (ASM) cells. The antiserum cross-reacted with protein bands of approximately 70, 120, and 150-160 kDa from the membranes of ASM cells, as well as heart sarcolemma. A cardiac Na(+)-Ca2+ exchanger cDNA probe hybridized to 7-kilobase (kb) mRNA from myocytes of the mesenteric artery. Thus ASM cells possess a "cardiac type" Na(+)-Ca2+ exchanger. The relative amounts of 7-kb mRNA and antigen detected on Northern and Western blots, respectively, however, indicate that vascular myocytes contain much less of this transporter than do cardiac myocytes. Immunofluorescence studies on cultured arterial myocytes suggest that the exchanger molecules are organized in reticular patterns over the cell surfaces. A similar pattern is observed when cells are stained for sarcoplasmic reticulum (SR) Ca(2+)-ATPase. This raises the possibility that the exchanger in the plasmalemma of arterial myocytes may be associated, perhaps functionally as well as structurally, with the underlying SR. The antiserum also cross-reacted with endothelial cell membranes, but labeling was lighter and more diffuse than in the myocytes.