RESUMO
The specific localization of L-type Ca(2+) channels in skeletal muscle triads is critical for their normal function in excitation-contraction (EC) coupling. Reconstitution of dysgenic myotubes with the skeletal muscle Ca(2+) channel alpha(1S) subunit restores Ca(2+) currents, EC coupling, and the normal localization of alpha(1S) in the triads. In contrast, expression of the neuronal alpha(1A) subunit gives rise to robust Ca(2+) currents but not to triad localization. To identify regions in the primary structure of alpha(1S) involved in the targeting of the Ca(2+) channel into the triads, chimeras of alpha(1S) and alpha(1A) were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the alpha(1S)/alpha(1A) chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of alpha(1A) were not incorporated into triads, chimeras containing the COOH terminus of alpha(1S) were correctly targeted. Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607-1661) proximal to the putative clipping site of alpha(1S). Transferring this triad targeting signal to alpha(1A) was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca(2+)-dependent EC coupling.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Músculo Esquelético/fisiologia , Sinais Direcionadores de Proteínas , Transporte Proteico , Potenciais de Ação , Sequência de Aminoácidos , Cálcio/metabolismo , Condutividade Elétrica , Junções Intercelulares/metabolismo , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Isoformas de Proteínas , Subunidades Proteicas , Proteínas Recombinantes de FusãoRESUMO
Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.
Assuntos
Canais de Cálcio/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio Tipo L , Linhagem Celular , Fusão de Membrana , Proteínas Musculares/genética , Músculo Esquelético/citologia , Músculo Esquelético/ultraestrutura , Ratos , Canal de Liberação de Cálcio do Receptor de RianodinaRESUMO
Rapid release of calcium from the sarcoplasmic reticulum (SR) of skeletal muscle fibers during excitation-contraction (e-c) coupling is initiated by the interaction of surface membrane calcium channels (dihydropyridine receptors; DHPRs) with the calcium release channels of the SR (ryanodine receptors; RyRs, or feet). We studied the early differentiation of calcium release units, which mediate this interaction, in BC3H1 cells. Immunofluorescence labelings of differentiating myocytes with antibodies against alpha1 and alpha2 subunits of DHPRs, RyRs, and triadin show that the skeletal isoforms of all four proteins are abundantly expressed upon differentiation, they appear concomitantly, and they are colocalized. The transverse tubular system is poorly organized, and thus clusters of e-c coupling proteins are predominantly located at the cell periphery. Freeze fracture analysis of the surface membrane reveals tetrads of large intramembrane particles, arranged in orderly arrays. These appear concomitantly with arrays of feet (RyRs) and with the appearance of DHPR/RyS clusters, confirming that the four components of the tetrads correspond to skeletal muscle DHPRs. The arrangement of tetrads and feet in developing junctions indicates that incorporation of DHPRs in junctional domains of the surface membrane proceeds gradually and is highly coordinated with the formation of RyR arrays. Within the arrays, tetrads are positioned at a spacing of twice the distance between the feet. The incorporation of individual DHPRs into tetrads occurs exclusively at positions corresponding to alternate feet, suggesting that the assembly of RyR arrays not only guides the assembly of tetrads but also determines their characteristic spacing in the junction.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Transporte , Proteínas Musculares/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L , Linhagem Celular , Membrana Celular/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Técnica de Fratura por Congelamento , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Camundongos , Microscopia Eletrônica , Músculo Esquelético/química , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestruturaRESUMO
We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the skeletal muscle dihydropyridine (DHP) receptor with immunofluorescence labeling of normal and dysgenic (mdg) muscle in culture. In normal myotubes both alpha subunits were localized in clusters associated with the T-tubule membranes of longitudinally as well as transversely oriented T-tubules. The DHP receptor-rich domains may represent the sites where triad junctions with the sarcoplasmic reticulum are being formed. In cultures from dysgenic muscle the alpha 1 subunit was undetectable and the distribution patterns of the alpha 2 subunit were abnormal. The alpha subunit did not form clusters nor was it discretely localized in the T-tubule system. Instead, alpha 2 was found diffusely distributed in parts of the T-system, in structures in the perinuclear region and in the plasma membrane. These results suggest that an interaction between the two alpha subunits is required for the normal distribution of the alpha 2 subunit in the T-tubule membranes. Spontaneous fusion of normal non-muscle cells with dysgenic myotubes resulted in a regional expression of the alpha 1 polypeptide near the foreign nuclei, thus defining the nuclear domain of a T-tubule membrane protein in multi-nucleated muscle cells. Furthermore, the normal intracellular distribution of the alpha 2 polypeptide was restored in domains containing a foreign "rescue" nucleus; this supports the idea that direct interactions between the DHP receptor alpha 1 and alpha 2 subunits are involved in the organization of the junctional T-tubule membranes.
Assuntos
Músculos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais , Especificidade de Anticorpos , Canais de Cálcio , Células Cultivadas , Imunofluorescência , Camundongos , Músculos/citologia , Músculos/embriologia , Ratos , Ratos Endogâmicos , Receptores Nicotínicos/químicaRESUMO
The type 3 ryanodine receptor (RyR3) is a ubiquitous calcium release channel that has recently been found in mammalian skeletal muscles. However, in contrast to the skeletal muscle isoform (RyR1), neither the subcellular distribution nor the physiological role of RyR3 are known. Here, we used isoform-specific antibodies to localize RyR3 in muscles of normal and RyR knockout mice. In normal hind limb and diaphragm muscles of young mice, RyR3 was expressed in all fibers where it was codistributed with RyR1 and with the skeletal muscle dihydropyridine receptor. This distribution pattern indicates that RyR3 is localized in the triadic junctions between the transverse tubules and the sarcoplasmic reticulum. During development, RyR3 expression declined rapidly in some fibers whereas other fibers maintained expression of RyR3 into adulthood. Comparing the distribution of RyR3-containing fibers with that of known fiber types did not show a direct correlation. Targeted deletion of the RyR1 or RyR3 gene resulted in the expected loss of the targeted isoform, but had no adverse effects on the expression and localization of the respective other RyR isoform. The localization of RyR3 in skeletal muscle triads, together with RyR1, is consistent with an accessory function of RyR3 in skeletal muscle excitation-contraction coupling.
Assuntos
Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Western Blotting , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L , ATPases Transportadoras de Cálcio/metabolismo , Diafragma/citologia , Diafragma/metabolismo , Regulação para Baixo , Imunofluorescência , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Membro Posterior/citologia , Membro Posterior/metabolismo , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Músculo Esquelético/ultraestrutura , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/enzimologia , Retículo Sarcoplasmático/metabolismoRESUMO
The biosynthesis of the peptides caerulein and PGLa in granular skin glands of Xenopus laevis proceeds through a pathway that involves discrete morphological rearrangements of the entire secretory compartment. Immunocytochemical localization of these peptides during gland development indicates that biosynthetic precursors are synthesized in intact secretory cells, whereas posttranslational processing requires morphological reorganization to a vacuolated stage. The bulk of the processed secretory material is then stored in vacuolae-derived storage granules. In the mature gland, storage granules are still formed at a low level. However, in this case processing takes place in a distinct cytoplasmic structure, the multicored body, which we suggest to be functionally equivalent to vacuolae. When granular glands regenerate after having lost all their storage granules upon strong stimuli, another morphological pathway is used. 2 wk after gland depletion, secretory cells become arranged in a monolayer that covers the luminal surface of the gland. Storage granules are formed continuously within these intact secretory cells. Here, precursor processing does not require a vacuolated stage as in newly generated glands but occurs in multicored bodies. Most storage granules seem to be formed in the third week of regeneration. The high biosynthetic activity is also reflected by the high activity of the putative processing enzyme dipeptidyl aminopeptidase during this period of regeneration.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Ceruletídeo/genética , Grânulos Citoplasmáticos/ultraestrutura , Peptídeos/genética , Glândulas Sebáceas/metabolismo , Pele/metabolismo , Animais , Ceruletídeo/biossíntese , Microscopia Eletrônica , Biossíntese Peptídica , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Regeneração , Glândulas Sebáceas/citologia , Glândulas Sebáceas/ultraestrutura , Pele/citologia , Pele/ultraestrutura , XenopusRESUMO
Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95-kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential-induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte , Microtúbulos/metabolismo , Proteínas Musculares/metabolismo , Músculos/metabolismo , Retículo Sarcoplasmático/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio/isolamento & purificação , Canais de Cálcio Tipo L , ATPases Transportadoras de Cálcio/isolamento & purificação , ATPases Transportadoras de Cálcio/metabolismo , Células Cultivadas , Imunofluorescência , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Camundongos , Camundongos Mutantes , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Dados de Sequência Molecular , Proteínas Musculares/isolamento & purificação , Músculos/citologia , Músculos/embriologia , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático/ultraestruturaRESUMO
The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Proteínas de Transporte/análise , Proteínas dos Microfilamentos , Proteínas Musculares/análise , Músculos/ultraestrutura , Miofibrilas/ultraestrutura , Sarcômeros/ultraestrutura , Animais , Anticorpos , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Peso Molecular , Músculos/química , Ratos , TropomodulinaRESUMO
We have used immunogold electron microscopy to study the organization of the acetylcholine receptor, 43 kd protein, voltage-sensitive Na+ channel, and ankyrin in the postsynaptic membrane of the rat neuromuscular junction. The acetylcholine receptor and the 43 kd protein are concentrated at the crests of the postsynaptic folds, coextensive with the subsynaptic density. In contrast, Na+ channels and ankyrin are concentrated in the membranes of the troughs and in perijunctional membranes, both characterized by discontinuous submembrane electron-dense plaques. This configuration of interspersed postsynaptic membrane domains enriched in either Na+ channels or acetylcholine receptors may facilitate the initiation of the muscle action potential. Furthermore, the results support the involvement of ankyrin in immobilizing Na+ channels in specific membrane domains, analogous to the proposed involvement of the 43 kd protein in acetylcholine receptor immobilization.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Membrana/metabolismo , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Canais de Sódio/ultraestrutura , Animais , Anquirinas , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/fisiologia , Diafragma/citologia , Diafragma/metabolismo , Imuno-Histoquímica , Proteínas de Membrana/análise , Proteínas de Membrana/fisiologia , Microscopia Eletrônica , Junção Neuromuscular/análise , Junção Neuromuscular/ultraestrutura , Ratos , Receptores Colinérgicos/análise , Canais de Sódio/metabolismo , Canais de Sódio/fisiologia , Membranas Sinápticas/análise , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestruturaRESUMO
We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the dihydropyridine (DHP) receptor and ankyrin in rat skeletal muscle with immunofluorescence and immunogold labeling techniques. All three proteins were concentrated in the triad junction formed between the T-tubules and sarcoplasmic reticulum. The alpha 1 and alpha 2 subunits of the DHP receptor were colocalized in the junctional T-tubule membrane, supporting their proposed association in a functional complex and the possible participation of the alpha 2 subunit in excitation-contraction coupling. Ankyrin label in the triad showed a distribution different from that of the DHP receptor subunits. In addition, ankyrin was found in longitudinally oriented structures outside the triad. Thus, ankyrin might be involved in organizing the triad and in immobilizing integral membrane proteins in T-tubules and the sarcoplasmic reticulum.
Assuntos
Proteínas Sanguíneas/análise , Proteínas de Membrana/análise , Músculos/análise , Receptores Nicotínicos/análise , Animais , Anquirinas , Canais de Cálcio , Imunofluorescência , Ouro , Técnicas Imunológicas , Microscopia Eletrônica , Músculos/ultraestrutura , Ratos , Distribuição TecidualRESUMO
Membrane depolarization inactivates acetylcholine receptor (AChR) genes in skeletal muscle. We have studied this process in C2C12 cells, focusing on the role of calcium. Cytoplasmic calcium was monitored with fluo-3, and the activity of receptor genes was measured with a sensitive transcript elongation assay. Removal of extracellular calcium or blockage of L-type calcium channels disrupts signaling, even when release of calcium from the sarcoplasmic reticulum (SR) is not impeded, whereas L channel agonists induce signaling without membrane depolarization or release of calcium from intracellular stores. Activators of calcium release from the SR do not inhibit AChR genes, either in C2C12 or in chicken skeletal muscle in vivo. It appears that calcium ions do not act as messengers between sarcolemma and nucleus but target a sensor near their port of entry where they initiate a signal that bypasses the SR.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Músculos/fisiologia , Retículo Sarcoplasmático/fisiologia , Transdução de Sinais , Transcrição Gênica , Potenciais de Ação , Animais , Linhagem Celular , Membrana Celular/fisiologia , Galinhas , Citoplasma/metabolismo , Estimulação Elétrica , Matriz Extracelular/metabolismo , Camundongos , Cloreto de Potássio/farmacologiaRESUMO
The relationship between the molecular composition and organization of the triad junction and the development of excitation-contraction (E-C) coupling was investigated in cultured skeletal muscle. Action potential-induced calcium transients develop concomitantly with the first expression of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR), which are colocalized in clusters from the time of their earliest appearance. These DHPR/RyR clusters correspond to junctional domains of the transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), respectively. Thus, at first contact T-tubules and SR form molecularly and structurally specialized membrane domains that support E-C coupling. The earliest T-tubule/SR junctions show structural characteristics of mature triads but are diverse in conformation and typically are formed before the extensive development of myofibrils. Whereas the initial formation of T-tubule/SR junctions is independent of association with myofibrils, the reorganization into proper triads occurs as junctions become associated with the border between the A band and the I band of the sarcomere. This final step in triad formation manifests itself in an increased density and uniformity of junctions in the cytoplasm, which in turn results in increased calcium release and reuptake rates.
Assuntos
Músculo Esquelético/embriologia , Potenciais de Ação , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L , Células Cultivadas , Imunofluorescência , Microscopia Eletrônica , Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático/fisiologia , Retículo Sarcoplasmático/ultraestrutura , Distribuição TecidualRESUMO
A full-length and a C-terminally truncated form of the calcium channel alpha(1S) subunit can be isolated from skeletal muscle. Here we studied whether full-length alpha(1S) is functionally incorporated into the skeletal muscle excitation-contraction coupling apparatus. A fusion protein of alpha(1S) with the green fluorescent protein attached to its C-terminus (alpha(1S)-GFP) or alpha(1S) and GFP separately (alpha(1S)+GFP) were expressed in dysgenic myotubes, which lack endogenous alpha(1S). Full-length alpha(1S)-GFP was targeted into triad junctions and restored calcium currents and excitation-contraction coupling. GFP remained colocalized with alpha(1S), indicating that intact alpha(1S)-GFP was inserted into triads and that the C-terminus remained associated with the excitation-contraction coupling apparatus.
Assuntos
Canais de Cálcio Tipo L/genética , Expressão Gênica , Músculo Esquelético/metabolismo , Transfecção , Canais de Cálcio Tipo L/deficiência , Canais de Cálcio Tipo L/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Condutividade Elétrica , Estimulação Elétrica , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Microtúbulos/metabolismo , Contração Muscular , Proteínas Recombinantes de Fusão , Retículo Sarcoplasmático/metabolismoRESUMO
Neurotransmitter release probability is related by high power to the local concentration of calcium in presynaptic terminals, which in turn is controlled by voltage-gated calcium channels. P/Q- and N-type channels trigger synaptic transmission in the majority of neurons of the central nervous system. However, whether and under which conditions both channel types act cooperatively or independently is still insufficiently understood. Previous studies suggested either a dominance of N- or P/Q-type channels, or a synergistic action of both channels, depending on the experimental paradigms. Thus, to provide insight into the properties of neurotransmitter release in cultured mouse hippocampal neurons, we used quantitative analysis of FM dye release from presynaptic boutons induced by high potassium membrane depolarization. Increasing extracellular potassium concentrations revealed a sigmoid dependence of FM dye release to the stimulation strength. Individual and combined application of the P/Q- and N-type channel-specific blockers ω-agatoxin-IVA and ω-conotoxin-GVIA, respectively, allowed us to specifically isolate the contribution of both channel types to release triggered with 40 mM KCl. Analysis of the release kinetics and the fractional release amplitude demonstrate that, whereas in only 15% of the synapses release depended exclusively on P/Q-type channels, the majority of synapses (85%) contained both N- and P/Q-type channels. Nevertheless, the kinetics of FM dye release in synapses containing both channel types was determined by the P/Q-type channels. Together, our data suggest a more direct coupling of P/Q-type channels to synaptic release compared to N-type channels, which may explain the high prevalence of neurological P/Q-type channelopathies.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Corantes Fluorescentes/farmacocinética , Hipocampo/citologia , Neurônios/citologia , Terminações Pré-Sinápticas/metabolismo , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Animais , Cloreto de Cádmio/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Cloreto de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxina GVIA/farmacologiaRESUMO
The importance and diversity of calcium signaling in the brain is mirrored by the expression of a multitude of voltage-activated calcium channel (Ca(V)) isoforms. Whereas the overall distributions of alpha(1) subunits are well established, the expression patterns of distinct channel isoforms in specific brain regions and neurons, as well as those of the auxiliary beta and alpha(2)delta subunits are still incompletely characterized. Further it is unknown whether neuronal differentiation and activity induce changes of Ca(V) subunit composition. Here we combined absolute and relative quantitative TaqMan reverse transcription PCR (RT-PCR) to analyze mRNA expression of all high voltage-activated Ca(V) alpha(1) subunits and all beta and alpha(2)delta subunits. This allowed for the first time the direct comparison of complete Ca(V) expression profiles of mouse cortex, hippocampus, cerebellum, and cultured hippocampal neurons. All brain regions expressed characteristic profiles of the full set of isoforms, except Ca(V)1.1 and Ca(V)1.4. In cortex development was accompanied by a general down regulation of alpha(1) and alpha(2)delta subunits and a shift from beta(1)/beta(3) to beta(2)/beta(4). The most abundant Ca(V) isoforms in cerebellum were Ca(V)2.1, beta(4), and alpha(2)delta-2, and in hippocampus Ca(V)2.3, beta(2), and alpha(2)delta-1. Interestingly, cultured hippocampal neurons also expressed the same Ca(V) complement as adult hippocampus. During differentiation specific Ca(V) isoforms experienced up- or down-regulation; however blocking electrical activity did not affect Ca(V) expression patterns. Correlation analysis of alpha(1), beta and alpha(2)delta subunit expression throughout all examined preparations revealed a strong preference of Ca(V)2.1 for beta(4) and alpha(2)delta-2 and vice versa, whereas the other alpha(1) isoforms were non-selectively expressed together with each of the other beta and alpha(2)delta isoforms. Together our results revealed a remarkably stable overall Ca(2+) channel complement as well as tissue specific differences in expression levels. Developmental changes are likely determined by an intrinsic program and not regulated by changes in neuronal activity.
Assuntos
Encéfalo/metabolismo , Canais de Cálcio/genética , Hipocampo/metabolismo , Envelhecimento/metabolismo , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Canais de Cálcio/química , Sinalização do Cálcio/genética , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Increased interest in the mechanism of excitation-contraction (E-C) coupling over the last few years has been accompanied by numerous investigations into the development of the underlying cellular structures. Areas of particular interest include: (1) the compartmentalization and specialization of an external and an internal membrane system, the T-tubules, and the sarcoplasmic reticulum, respectively; (2) interactions between the membrane proteins of both systems upon the formation of a junction, the triad; and (3) membrane-cytoskeletal interactions leading to the orderly arrangement of the triads with respect to the myofibrils. Structural studies using newly available specific molecular probes and a variety of in vivo and in vitro model systems have provided new insights into the cellular and molecular mechanisms involved in the development of the E-C coupling apparatus in skeletal muscle.
Assuntos
Músculos/embriologia , Retículo Sarcoplasmático/ultraestrutura , Animais , Contração Muscular , Músculos/ultraestrutura , Organelas/ultraestrutura , RatosRESUMO
We have investigated the onset and maturation of action potential- and calcium-induced calcium release from the sarcoplasmic reticulum during the differentiation of excitation-contraction coupling in skeletal muscle. Microfluorometry and video imaging of cultured myotubes loaded with the fluorescent calcium indicator fluo-3 revealed the dynamics, time course, and physiological properties of calcium transients as well as their changes during development. Spontaneous and stimulated contractions in well-differentiated myotubes are accompanied by brief (200-500 ms) increases in the concentration of free cytoplasmic calcium. These transients are modulated by sub-threshold concentrations of caffeine, resulting in a plateau of elevated calcium. Two novel types of calcium transients were observed in non-contracting myotubes. 1) Fast localized transients (FLTs) are radially restricted focal release events that occur spontaneously within the myoplasm at various densities and frequencies. 2) Upon addition of caffeine, propagating calcium waves are generated (35-70 microns/s velocity), which are accompanied by contractures. Aside from caffeine sensitivity, calcium waves and contraction-related sustained release events are similar in amplitude and duration, as well as in their inactivation and refractory properties. Thus, these transients may represent calcium-induced calcium release in quiescent and active myotubes, respectively. Following one calcium-induced calcium release event, myotubes become refractory to new calcium-induced transients; however, action potential-induced transients and FLTs are not blocked. This suggests that these transients occur by distinct release mechanisms and that dual modes of calcium release exist prior to the coupling of calcium release to excitation.
Assuntos
Potenciais de Ação , Cálcio/metabolismo , Músculos/metabolismo , Sistemas do Segundo Mensageiro , Animais , Transporte Biológico/efeitos dos fármacos , Cafeína/farmacologia , Cálcio/farmacologia , Diferenciação Celular , Embrião de Galinha , Camundongos , Contração Muscular/efeitos dos fármacos , Músculos/citologia , Músculos/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Retículo Sarcoplasmático/metabolismoRESUMO
During excitation-contraction (e-c) coupling of striated muscle, depolarization of the surface membrane is converted into Ca2+ release from internal stores. This process occurs at intracellular junctions characterized by a specialized composition and structural organization of membrane proteins. The coordinated arrangement of the two key junctional components--the dihydropyridine receptor (DHPR) in the surface membrane and the ryanodine receptor (RyR) in the sarcoplasmic reticulum--is essential for their normal, tissue-specific function in e-c coupling. The mechanisms involved in the formation of the junctions and a potential participation of DHPRs and RyRs in this process have been subject of intensive studies over the past 5 years. In this review we discuss recent advances in understanding the organization of these molecules in skeletal and cardiac muscle, as well as their concurrent and independent assembly during development of normal and mutant muscle. From this information we derive a model for the assembly of the junctions and the establishment of the precise structural relationship between DHPRs and RyRs that underlies their interaction in e-c coupling.
Assuntos
Canais de Cálcio/fisiologia , Coração/fisiologia , Modelos Biológicos , Contração Muscular , Músculo Esquelético/fisiologia , Contração Miocárdica , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L , Membrana Celular/fisiologia , Membranas Intracelulares/fisiologia , Proteínas Musculares/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático/fisiologia , Transdução de SinaisRESUMO
Distribution and association of neurotensin (NT)- and xenopsin (XP)-like peptides were investigated using immunocytochemical techniques in the amphibian gut. Antisera against both groups of peptides showed an identical distribution pattern of NT- and XP-positive cells in Xenopus laevis gastrointestinal tract. Immunolabeling of consecutive semithin sections revealed the coexistence of NT- and XP-like substances within cells of the stomach and small intestine. Recent reports of the colocalization of XP-like material with gastrin in mammalian G cells led us to study the association of NT/XP-like peptides with members of the gastrin/cholecystokinin (CCK)/caerulein (G/C) family in amphibians. The data obtained from immunolabeling serial sections with NT/XP-specific and G/C-specific antisera show that in some intestine NT/XP- and G/C-like peptides do exist in the same cells. In the stomach, however, G/C-like material is confined to endocrine cells of the antral region, while NT/XP-like substances occur in distinct cells accumulating in cardial glands but absent in the pyloric glands. Our findings thus indicate that in amphibian gastrointestinal tract there is some association between the regulatory peptide families NT/XP and G/C, similar to mammals. The regional distribution of both hormone families, however, is different from that in mammals.
Assuntos
Sistema Digestório/análise , Neuropeptídeos/análise , Proteínas de Xenopus , Animais , Ceruletídeo/análise , Colecistocinina/análise , Gastrinas/análise , Técnicas Imunológicas , Neurotensina/análise , Oligopeptídeos/análise , Pentagastrina/análise , Peptídeos , Xenopus laevisRESUMO
The formation and maintenance of the highly regular organization of membrane systems and proteins in striated muscle require specific membrane-membrane and membrane-cytoskeleton interactions. The development of T-tubules and sarcoplasmic reticulum (SR) was followed in gastrocnemius muscle fibers from chicken embryos between 12 days (E12) and 21 days (E21) of incubation, with particular attention to their relationship with one another and with the myofibrils. The fluorescent lipid analog DiIC16[3] was used to label either the external membranes (plasmalemma and transverse (T)-tubules) or the internal SR in living and fixed muscle. Short membrane invaginations can first be seen in fibers at E14, and at E15 longitudinal T-tubules appear in the periphery of the fibers. A complex network of T-tubules filling the whole fiber diameter develops suddenly at E16. In contrast, SR is abundant at the earliest observed stage (E12) and forms regularly spaced cross striations located at the I-Z-I bands. These correspond to a specific accumulation of smooth membranes around the Z-discs seen in electron micrographs. While SR is specifically associated with the newly formed myofibrils in the periphery of the fibers, the disposition of early T-tubules shows little specific relationship to either SR or the myofibrils. However, electron microscopy shows that junctions between T-tubules and SR are formed during this period (Takekura and Franzini-Armstrong, submitted for publication). Junctions do not acquire a specific relation to the myofibrils until around hatching when triads begin to reorganize into their mature location, the A-I junction. These findings indicate three key events in the organization of T-tubules and SR in the sarcomeres: (1) early SR/Z-line interactions independent of T-tubules; (2) SR/T-tubule interactions to form the triad junctions, independent from the myofibrils; and (3) the late association of the junctional complexes with the myofibrils at the A-I border.