RESUMEN
The skeletal muscle L-type Ca(2+) channel or dihydropyridine(DHP)-sensitive receptor is a key molecule involved in membrane voltage-sensing, sarcoplasmic reticulum Ca(2+) release, and muscle contraction. Previous work from our laboratory has shown that the insulin-like growth factor-1 (IGF-1) increases skeletal muscle L-type Ca(2+) channel or dihydropyridine-sensitive receptor DHPRalpha(1S) transcriptional activity by acting on the cyclic AMP response element binding protein (CREB) element of the promoter region; however, the cellular signaling mediating this process is not known. In this study, we investigated the signaling pathway whereby IGF-1 enhances the expression of DHPRalpha(1S) in C2C12 myotubes, using a molecular, pharmacological and electrophysiological approach. We found that inhibition of the Ca(2+)/Calmodulin (CaM)-dependent protein kinase or calcineurin, influenced IGF-1-induced increase in DHPRalpha(1S) expression, as detected by recording the luminescence of the DHPRalpha(1S) promoter-luciferase fusion construct and by immunoblot analysis of the DHPR alpha1 subunit. IGF-1 significantly increased CaM kinase and calcineurin activity and the cellular levels of phosphorylated CREB in a time-dependent manner. The role of CaM kinase and calcineurin in DHPRalpha(1S) expression was confirmed by functional recording of the effects of the inhibition of the kinase and phosphatase on IGF-1-mediated enhancement of charge movement. These results support the conclusion that IGF-1 controls CREB phosphorylation by activating a phosphorylation and dephosphorylation cascade, which ultimately modulates the DHPRalpha(1S) gene transcription.
Asunto(s)
Calcineurina/metabolismo , Canales de Calcio Tipo L/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Esquelético/metabolismo , Transcripción Genética , Animales , Canales de Calcio Tipo L/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Luciferasas , Ratones , Regiones Promotoras Genéticas , Proteínas Recombinantes , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
In the present work, we studied the recovery from fatigue (RF) of single intact fast- and slow-twitch muscle fibers from young (age 5--7 months) and old (age 22--24 months) mice. To examine whether differences in RF underlie decreases in muscle strength and endurance with aging, we performed in vitro experiments in manually dissected extensor digitorum longus (EDL) and soleus muscle fibers. We measured the recovery of the maximum force every 5 min for a total period of 30 min after inducing fiber fatigue. Fibers were classified, according to the fatigue index, into the following three groups: 0.75--0.99, 0.5--0.74, and <0.5. Although the tetanic tension of EDL and soleus fibers from young and old mice recovered significantly, no statistically significant difference in tension or recovery time was observed between age groups. These data support the concept that the reported decline in muscle force and endurance with aging is not related to changes in RF of individual muscles fibers.
Asunto(s)
Envejecimiento/fisiología , Fatiga Muscular/fisiología , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Animales , Ratones , Ratones Endogámicos DBA , Contracción Muscular/fisiología , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Tiempo de Reacción/fisiologíaRESUMEN
In the present work, we investigate age-dependent changes in isometric endurance in response to repetitive stimulation in single intact fast- and slow-twitch muscle fibers from young and old mice. To examine this issue we performed in vitro experiments in manually dissected EDL and soleus muscle fibers. We examined the force generation capacity of fibers in response to two stimulation protocols characterized by different inter-tetanic intervals, named short (1-s) and long interval (3.65-s). Fatigability was measured according to the fatigue index (FI, ratio between the maximum tension recorded in the last over the first tetanus in a train of pulses), the time course of the FI and sag (gradual decrease in force during a partially fused tetanic contraction). Fibers were classified according to the FI using two different criteria previously used in the literature (first criterion: FI > or = 1, 075-099, 0.5-074 and < 0.5; second criterion: FI > or = 1, 0.75-0.99, 0.25-0.74 and < 0.25). The fatigue index distribution recorded in the population of fibers corresponding to EDL and soleus muscles from young and old mice studied with the short and long interval protocols was not statistically different. In summary, these results support the concept that the decline in mechanical performance with aging is not related with changes in fatigability of individual fast- or slow twitch muscles fibers.
Asunto(s)
Envejecimiento/fisiología , Fatiga Muscular/fisiología , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/fisiología , Animales , Ratones , Ratones Endogámicos DBA , Factores de TiempoRESUMEN
In the present work, we investigated whether IGF-1 regulates the transcription of the genes encoding the L-type Ca2+ channel (DHPR) channel and RyR1 in young adult and senescent mice. To this end, a transgenic mouse model overexpressing IGF-1 exclusively in skeletal muscle (S1S2) was studied at different ages and the results were compared with wild type age-matched mice (FVB). We found that ribosomal RNA expression did not change significantly either with age or IGF-1 according to ribonuclease protection and nuclear run-on transcription assays. Transgenic overexpression of IGF-1 resulted in marked increases in skeletal muscle DHPR alpha(1S) and RyR1 mRNA in young and old mice and in enhanced DHPR alpha(1S) nuclear transcription in skeletal muscles from young mice when normalized to 28S ribosomal RNA. These results support the concept that IGF-1 regulates the expression of DHPR by modulating DHPR alpha(1S) nuclear transcription.
Asunto(s)
Envejecimiento , Canales de Calcio Tipo L/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Esquelético/metabolismo , ARN Mensajero/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Transcripción Genética/fisiología , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Southern Blotting , Canales de Calcio Tipo L/metabolismo , Sondas de ADN , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Ratones Transgénicos , Músculo Esquelético/citología , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Excitation-contraction (EC) uncoupling is a primary muscle alteration and constitutes a major cause of decline in skeletal muscle force with aging. The structural substratum for EC uncoupling in muscles from aging mammals is a reduction in number of dihydropyridine receptors (DHPR) at the T-tubule and SR membrane leading to an increase in the percent of sarcoplasmic reticulum calcium release channels or ryanodine receptors (RyR1) uncoupled to DHPR. The main functional consequence of this alteration is a failure in the transduction of sarcolemmal depolarization into a calcium signal and a mechanical response. This review summarizes recent studies from our laboratory aimed at elucidating the modulation of EC coupling by insulin-like growth factor-1 (IGF-1) in skeletal muscle. We demonstrated that transgenic overexpression of human IGF-1 exclusively in skeletal muscle increases the number and prevents age-related decline in the number of DHPR. IGF-1 enhances rat skeletal muscle DHPR function and gene expression. The functional significance of these findings is that IGF-1 prevents the age-related decline in muscle force.
Asunto(s)
Envejecimiento/fisiología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Animales , Canales de Calcio Tipo L/fisiología , Proteínas Musculares/fisiología , Músculo Esquelético/metabolismo , Atrofia Muscular/prevención & control , Ratas , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
In this work we tested the hypothesis that skeletal muscle fibers from aging mice exhibit a significant decline in myoplasmic Ca(2+) concentration resulting from a reduction in L-type Ca(2+) channel (dihydropyridine receptor, DHPR) charge movement. Skeletal muscle fibers from the flexor digitorum brevis (FDB) muscle were obtained from 5-7-, 14-18-, or 21-24-month-old FVB mice and voltage-clamped in the whole-cell configuration of the patch-clamp technique according to described procedures (Wang, Z.-M., M. L. Messi, and O. Delbono. 1999. Biophys. J. 77:2709-2716). Total charge movement or the DHPR charge movement was measured simultaneously with intracellular Ca(2+) concentration. The maximum charge movement (Q(max)) recorded (mean +/- SEM, in nC microF(-1)) was 53 +/- 3.2 (n = 47), 51 +/- 3.2 (n = 35) (non-significant, ns), and 33 +/- 1.9 (n = 32) (p < 0.01), for the three age groups, respectively. Q(max) corresponding to the DHPR was 43 +/- 3.3, 38 +/- 4.1 (ns), and 25 +/- 3.4 (p < 0.01) for the three age groups, respectively. The peak intracellular [Ca(2+)] recorded at 40 mV (in microM) was 15.7 +/- 0. 12, 16.7 +/- 0.18 (ns), and 8.2 +/- 0.07 (p < 0.01) for the three age groups, respectively. No significant changes in the voltage distribution or steepness of the Q-V or [Ca(2+)]-V relationship were found. These data support the concept that the reduction in the peak intracellular [Ca(2+)] results from a larger number of ryanodine receptors uncoupled to DHPRs in skeletal muscle fibers from aging mammals.
Asunto(s)
Envejecimiento/metabolismo , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Músculo Esquelético/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Electrofisiología , Técnicas In Vitro , Líquido Intracelular/metabolismo , Potenciales de la Membrana , Ratones , Técnicas de Placa-ClampRESUMEN
In the present study we measured, for the first time, the isometric specific force (SF, force normalized to cross sectional area) generated by single intact fibers from fast- (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from young adult (2-6), middle-aged (12-14) and old (20-24 month-old) mice. SF has also been measured in single intact flexor digitorum brevis fibers from young mice. Muscle fibers have been classified into fast- or slow-twitch based on the contraction kinetics. Maximum SF recorded in EDL and soleus fibers from young and middle-aged mice did not differ significantly. A significant age-dependent decline in maximum SF was recorded in EDL and soleus fibers from young or middle-aged to old mice. The SF was 377 +/- 18, 417 +/- 20 and 279 +/- 18 kPa for EDL fibers from young, middle-aged and old mice, respectively and 397 +/- 17, 405 +/- 24 and 320 +/- 33 kPa for soleus fibers from age-matched mice, respectively. The frequency needed to elicit maximum force in EDL and soleus fibers from middle-aged to old mice did not differ significantly. In conclusion, the specific force developed by both fast and slow-twitch single intact muscle fibers declines with aging and more significantly in the former.
Asunto(s)
Envejecimiento/fisiología , Contracción Muscular , Desarrollo de Músculos , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/crecimiento & desarrollo , Animales , Femenino , Contracción Isométrica , Cinética , Masculino , Ratones , Ratones Endogámicos DBA , Fibras Musculares de Contracción Rápida/química , Fibras Musculares de Contracción Lenta/química , Músculo Esquelético/fisiología , Cadenas Pesadas de Miosina/análisis , Isoformas de Proteínas/análisis , Organismos Libres de Patógenos EspecíficosRESUMEN
Primary skeletal muscle cells were cultured in a normal- (1.8 mM) or high- (4.8 mM) Ca2+ culture medium to determine whether Ca2+ modulates the number of L-type Ca2+ channels. Skeletal myoballs cultured in a normal medium showed, when exposed to a high extracellular [Ca2+], ([Ca2+]e) a transient increase in intracellular [Ca2+] ([Ca2+]i) from a resting concentration of 60 to 160 nM. By day 3, however, when the experiments were made, [Ca2+]i no longer differed from control (pre-exposure to high Ca2+). The maximum charge movements in myoballs incubated in 1.8 and 4.8 mM were 16.4+/-1.05 (n=56) and 24.1+/-1.18 nC/microF (n=58; P<0.01), respectively, and peak Ca2+ currents at 20 mV were -10.8+/-1.09 (n=46) and -12.8+/-0.75 nA/microF (n=82), respectively (P>0.05). The tail current amplitudes in 1.8 and 4.8 mM Ca2+-treated cells were -9.3+/-1.23 and -14.2+/-1.37 nA/microF (P<0.05), respectively, at 10 mV and -15.3+/-1.76 and -23.6+/-2.02 nA/microF (P<0.05), respectively at 60 mV. The maximum binding of [3H]PN200-110 (a radioligand specific for L-type Ca2+ channel alpha1 subunits) in myoballs cultured in 1.8 and 4.8 mM [Ca2+]e was 1.34+/-0.23 and 3.2+/-0.63 pmol/mg protein (n=8; P<0.02), respectively. The increase in [Ca2+]i associated with the increases in charge movements, tail currents and the number of L-type Ca2+ channel alpha1 subunits in skeletal muscle cells cultured in high [Ca2+]e support the concept that extracellular Ca2+ influx modulates the expression of L-type Ca2+ channels in skeletal muscle cells.
Asunto(s)
Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/fisiología , Calcio/farmacología , Músculo Esquelético/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Células Cultivadas , Conductividad Eléctrica , Isradipino/metabolismo , Potenciales de la Membrana , Ratas , Ratas Sprague-DawleyRESUMEN
Intramembrane charge movement (Q), Ca(2+) conductance (G(m)) through the dihydropyridine-sensitive L-type Ca(2+) channel (DHPR) and intracellular Ca(2+) fluorescence (F) have been recorded simultaneously in flexor digitorum brevis muscle fibers of adult mice, using the whole-cell configuration of the patch-clamp technique. The voltage distribution of Q was fitted to a Boltzmann equation; the Q(max), V(1/2Q), and effective valence (z(Q)) values were 41 +/- 3.1 nC/microF, -17.6 +/- 0.7 mV, and 2.0 +/- 0.12, respectively. V(1/2G) and z(G) values were -0.3 +/- 0.06 mV and 5.6 +/- 0.34, respectively. Peak Ca(2+) transients did not change significantly after 30 min of recording. F was fit to a Boltzmann equation, and the values for V(F1/2) and z(F) were 6.2 +/- 0.04 mV and 2.4, respectively. F was adequately fit to the fourth power of Q. These results demonstrate that the patch-clamp technique is appropriate for recording Q, G(m), and intracellular [Ca(2+)] simultaneously in mature skeletal muscle fibers and that the voltage distribution of the changes in intracellular Ca(2+) can be predicted by a Hodgkin-Huxley model.
Asunto(s)
Calcio/metabolismo , Conductividad Eléctrica , Fibras Musculares Esqueléticas/metabolismo , Técnicas de Placa-Clamp/métodos , Animales , Canales de Calcio Tipo L/metabolismo , Membrana Celular/metabolismo , Pie , Espacio Intracelular/metabolismo , Ratones , Fibras Musculares Esqueléticas/citología , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
1. We investigated whether insulin-like growth factor-1 (IGF-1), an endogenous potent activator of skeletal muscle proliferation and differentiation, enhances L-type Ca2+ channel gene expression resulting in increased functional voltage sensors in single skeletal muscle cells. 2. Charge movement and inward Ca2+ current were recorded in primary cultured rat myoballs using the whole-cell configuration of the patch-clamp technique. Ca2+ current and maximum charge movement (Qmax) were potentiated in cells treated with IGF-1 without significant changes in their voltage dependence. Peak Ca2+ current in control and IGF-1-treated cells was -7.8 +/- 0.44 and -10. 5 +/- 0.37 pA pF-1, respectively (P < 0.01), whilst Qmax was 12.9 +/- 0.4 and 22.0 +/- 0.3 nC microF-1, respectively (P < 0.01). 3. The number of L-type Ca2+ channels was found to increase in the same preparation. The maximum binding capacity (Bmax) of the high-affinity radioligand [3H]PN200-110 in control and IGF-1-treated cells was 1.21 +/- 0.25 and 3.15 +/- 0.5 pmol (mg protein)-1, respectively (P < 0.01). No significant change in the dissociation constant for [3H]PN200-110 was found. 4. Antisense RNA amplification showed a significant increase in the level of mRNA encoding the L-type Ca2+ channel alpha1-subunit in IGF-1-treated cells. 5. This study demonstrates that IGF-1 regulates charge movement and the level of L-type Ca2+ channel alpha1-subunits through activation of gene expression in skeletal muscle cells.
Asunto(s)
Canales de Calcio/biosíntesis , Canales de Calcio/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Músculo Esquelético/metabolismo , Animales , Canales de Calcio Tipo L , Estimulación Eléctrica , Electrofisiología , Isradipino/farmacología , Potenciales de la Membrana/fisiología , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Técnicas de Placa-Clamp , ARN sin Sentido , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-DawleyRESUMEN
The dihydropyridine receptor (DHPR) and ryanodine receptor (RYR1) are needed for excitation-contraction coupling in skeletal muscle. Previous studies from this laboratory have shown DHPR-RYR1 uncoupling in 33-month-old Fischer 344 x Brown Norway F1 (F344BNF1) rats fed ad libitum. The purpose of the present study is to determine whether caloric restriction prevents age-related impairments in skeletal muscle function and expression of DHPR and RyR1. Bundles of soleus and extensor digitorum longus (EDL) were studied from rats fed ad libitum and on 60 percent caloric restriction. Significant differences were found in peak twitch or tetanic tension between the ad libitum and calorie-restricted groups in soleus and EDL muscles. A significant increase in the expression of DHPR and RyR1 was observed in caloric restricted rats. These results show that calorie restriction preserves the mechanical properties of aging hind-limb skeletal muscle and maintains the level of DHPR and RyR1 in aged F344BNF1 rats fed ad libitum.
Asunto(s)
Envejecimiento/fisiología , Ingestión de Energía/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Animales , Canales de Calcio/análisis , Canales de Calcio/metabolismo , Canales de Calcio Tipo L , Cruzamientos Genéticos , Privación de Alimentos/fisiología , Miembro Posterior , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/análisis , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344 , Canal Liberador de Calcio Receptor de Rianodina/análisis , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Excitation-contraction uncoupling has been identified as a mechanism underlying skeletal muscle weakness in aging mammals (sarcopenia). The basic mechanism for excitation-contraction uncoupling is a larger number of ryanodine receptors (RyR1) uncoupled to dihydropyridine receptors (DHPRs) (Delbono, O., O'Rourke, K. S., and Ettinger, W. H. (1995) J. Membr. Biol. 148, 211-222). In the present study, we used transgenic mice overexpressing human insulin-like growth factor-1 exclusively in skeletal muscle to test the hypothesis that a high concentration of IGF-1 prevents age-related decreases in DHPR number and in muscle force. Transgenic mice express 10-20-fold higher IGF-1 concentrations than nontransgenic mice at all ages (1-24 months). The number of DHPRs is 50-100% higher, and the DHPR/RyR1 ratio is 40% higher in transgenic soleus (predominantly type I fiber muscles), extensor digitorum longus (predominantly type II fiber muscles), and the pool of type I and type II fiber muscles than in nontransgenic young (6 months), adult (12 months), and old (24 months) mice. Furthermore, no age-related changes in DHPRs and the DHPR/RyR1 ratio were observed in transgenic muscles. The specific single twitch and tetanic muscle force in old transgenic soleus and extensor digitorum longus muscles are 50% higher than in old nontransgenic muscles. Taken together, these results support the concept that IGF-1- dependent prevention of age-related decline in DHPR expression is associated with stronger muscle contraction in older transgenic mice.
Asunto(s)
Envejecimiento/metabolismo , Canales de Calcio/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Esquelético/metabolismo , Animales , Canales de Calcio Tipo L , Humanos , Ratones , Ratones Transgénicos , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
The dihydropyridine receptor (DHPR), a voltage-gated L-type Ca2+ channel, and the Ca2+ release channel/ryanodine receptor isoform-1 (RyR1) are key molecules involved in skeletal muscle excitation-contraction coupling. We have reported age-related decreases in the level of DHPR expression in fast- and slow-twitch muscles from Fisher 344 cross Brown Norway (F344BNX) rats (Renganathan, Messi and Delbono, J. Membr. Biol. 157 (1997) 247-253). Based on these studies we postulate that excitation-contraction uncoupling is a basic mechanism for the decline in muscle force with aging (Delbono, Renganathan and Messi, Muscle Nerve Suppl. 5 (1997) S88-92). In the present study, we extended our studies to older ages and we intended to prevent or retard excitation-contraction uncoupling by restricting the caloric intake of the F344BNX rats from 16 weeks of age. Three age groups, 8-, 18-, and 33-month old caloric restricted rats, were compared with ad libitum fed animals. The number of DHPR and RyR1 and DHPR/RyR1 ratio (an index of the level of receptors uncoupling) in skeletal muscles of 8-month and 18-month rats was not significantly different in either ad libitum fed or caloric restricted rats. However, the age-related decrease in the number of DHPR, RyR1 and DHPR/RyR1 ratio observed in 33-month old ad libitum fed rats was absent in 33-month old caloric restricted rats. These results suggest that caloric restriction prevents age-related decreases in the number of DHPR, RyR1 and DHPR/RyR1 ratio observed in fast- and slow-twitch rat skeletal muscles.
Asunto(s)
Envejecimiento/metabolismo , Canales de Calcio/metabolismo , Ingestión de Energía , Músculo Esquelético/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Análisis de Varianza , Animales , Canales de Calcio Tipo L , Femenino , Ratas , Ratas Endogámicas F344RESUMEN
This study examined the binding and functional properties of human alpha7 neuronal nicotinic acetylcholine receptors stably expressed in human embryonic kidney (HEK) 293 cells following chronic treatment with nicotinic receptor ligands. Treatment of cells with (-)-nicotine (100 microM) for 120 h increased the Bmax values of [125I]alpha-bungarotoxin binding 2.5-fold over untreated cells. This effect was concentration-dependent (EC50) = 970 microM) and a 6-fold upregulation was observed with the maximal concentration of (-)-nicotine tested. Also, treatment of cells with ligands of varying intrinsic activities including (+/-)-epibatidine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 1,1-dimethyl-4-phenyl piperazinium iodide (DMPP) also upregulated [125I]alpha-bungarotoxin binding. A concentration-dependent upregulation of binding sites was also observed following treatment with the alpha7 nicotinic receptor antagonist, methyllycaconitine (EC50 = 92 microM) with a maximal upregulation of about 7-fold. Functionally, the peak amplitude of the whole-cell currents recorded by fast application of (-)-nicotine after chronic treatment of cells with concentrations of (-)-nicotine (1000 microM) or methyllycaconitine (10 microM) that elicited similar increases in binding levels (3.5-fold) resulted in increases of 2-fold (505 +/- 21 pA) and 6-fold (1820 +/- 137 pA) respectively in whole cell current amplitude compared to untreated cells (267 +/- 24 pA). These studies clearly demonstrate that long-term exposure to both activator and antagonist ligands can increase the density of alpha7 nicotinic receptors and can differentially enhance nicotinic receptor function.
Asunto(s)
Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiología , Aconitina/análogos & derivados , Aconitina/farmacología , Compuestos de Bencilideno/farmacología , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Bungarotoxinas/metabolismo , Bungarotoxinas/farmacología , Yoduro de Dimetilfenilpiperazina/farmacología , Humanos , Insecticidas/farmacología , Radioisótopos de Yodo , Riñón/efectos de los fármacos , Riñón/ultraestructura , Cinética , Ligandos , Nicotina/farmacología , Antagonistas Nicotínicos/metabolismo , Piridinas/farmacología , Receptores Nicotínicos/metabolismo , Transfección , Regulación hacia Arriba/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The number of dihydropyridine receptors (DHPR) and sarcoplasmic reticulum (SR) Ca2+ release channels (RyR1) and their interaction determine the efficacy of the sarcolemmal excitation-SR Ca2+ release-contraction coupling (ECC). Both receptors play a central role in ECC as demonstrated in various animal species and muscle subtypes. In the present work we studied the effect of transgenic overexpression of human insulin-like growth factor 1 (hIGF-1) on the levels of these two Ca2+ channels in extensor digitorum longus (EDL) (fast-twitch), soleus (slow-twitch) and pool of fast- and slow-twitch muscles from adult C57BL/6 mice. Muscles from hIGF-1 transgenic mice showed a significant increase in IGF-1 concentration (20-30-fold) and in the number of DHPR (52% increase) whereas no significant change in RyR1 binding sites was detected. The differential effect on DHPR and RyR1 resulted in a 30% increase in DHPR/RyR1 ratio. Fast- and slow-twitch muscles showed 50 and 70% increase in the number of DHPR and 30 and 80% increase in DHPR/RyR1, respectively. These results support the concept that the increased autocrine/paracrine secretion of hIGF-1 exerts potent stimulatory effects on DHPR alpha1 subunit expression in adult skeletal muscle.
Asunto(s)
Canales de Calcio/metabolismo , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Músculo Esquelético/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Canales de Calcio Tipo L , Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
We investigated the modulation of the skeletal muscle L-type Ca2+ channel/dihydropyridine receptor in response to insulin-like growth factor-1 receptor (IGF-1R) activation in single extensor digitorum longus muscle fibers from adult C57BL/6 mice. The L-type Ca2+ channel activity in its dual role as a voltage sensor and a selective Ca2+-conducting pore was recorded in voltage-clamp conditions. Peak Ca2+ current amplitude consistently increased after exposure to 20 ng/ml IGF-1 (EC50 = 5.6 +/- 1.8 nM). Peak IGF-1 effect on current amplitude at -20 mV was 210 +/- 18% of the control. Ca2+ current potentiation resulted from a shift in 13 mV of the Ca2+ current-voltage relationship toward more negative potentials. The IGF-1-induced facilitation of the Ca2+ current was not associated with an effect on charge movement amplitude and/or voltage distribution. These phenomena suggest that the L-type Ca2+ channel structures involved in voltage sensing are not involved in the response to the growth factor. The modulatory effect of IGF-1 on L-type Ca2+ channel was blocked by tyrosine kinase and PKC inhibitors, but not by a cAMP-dependent protein kinase inhibitor. IGF-1-dependent phosphorylation of the L-type Ca2+ channel alpha1 subunit was demonstrated by incorporation of [gamma-32P]ATP to monolayers of adult fast-twitch skeletal muscles. IGF-1 induced phosphorylation of a protein at the 165 kDa band, corresponding to the L-type Ca2+ channel alpha1 subunit. These results show that the activation of the IGF-1R facilitates skeletal muscle L-type Ca2+ channel activity via a PKC-dependent phosphorylation mechanism.
Asunto(s)
Canales de Calcio/metabolismo , Músculo Esquelético/metabolismo , Receptor IGF Tipo 1/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Canales de Calcio Tipo L , Dihidropiridinas/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Fosforilación , Proteína Quinasa C/metabolismoRESUMEN
Spinal cord motoneurons (MNs) undergo a process of cell death during embryonic development and are the target of lethal acquired or inherited disorders, such as the amyotrophic lateral sclerosis. Therefore, the identification of mechanisms leading to MN survival is of crucial importance. Elevations in intracellular Ca2+ promote chicken MN survival during the embryonic period of naturally occurring cell death. We have recently demonstrated that the alpha7 nicotinic acetylcholine receptor (nAChR) mediates significant increases in free Ca2+ concentration at membrane potentials at which other pathways for Ca2+ influx are inactive. Although it is possible that Ca2+ influx through alpha7 nAChR promotes cell survival, the relation between alpha7 nAChR activation, cytosolic free Ca2+ and mammalian spinal cord MN survival has not been established. In the present study we have now demonstrated that Ca2+ influx through the alpha7-subunit is sufficient to rescue a significant number of cultured spinal cord MNs from programmed cell death induced by trophic factor deprivation. This is the first demonstration that neuronal nAChRs are involved in the regulation of MN survival.
Asunto(s)
Neuronas Motoras/fisiología , Receptores Nicotínicos/metabolismo , Médula Espinal/citología , Acetilcolina/farmacología , Aconitina/análogos & derivados , Aconitina/farmacología , Animales , Calcio/metabolismo , Supervivencia Celular , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Nicotina/farmacología , RatasRESUMEN
The mechanisms underlying skeletal muscle functional impairment and structural changes with advanced age are only partially understood. In the present study, we support and expand our theory about alterations in sarcolemmal excitation-sarcoplasmic reticulum Ca2+ release-contraction uncoupling as a primary skeletal muscle alteration and major determinant of weakness and fatigue in mammalian species including humans. To test the hypothesis that the number of RYR1 (ryanodine receptor) uncoupled to DHPR (dihydropyridine receptor) increases with age, we performed high-affinity ligand binding studies in soleus, extensor digitorum longus (EDL) and in a pool of several skeletal muscles consisting of a mixture of fast- and slow-twitch muscle fibers in middle-aged (14-month) and old (28-months) Fisher 344 Brown Norway F1 hybrids rats. The number of DHPR, RYR1, the coupling between both receptors expressed as the DHPR/RYR1 maximum binding capacity, and their dissociation constant for high-affinity ligands were measured. The DHPR/RYR1 ratio was significantly reduced in the three groups of muscles (pool: 1.03 +/- 0.15 and 0.80 +/- 0.11, soleus: 0.44 +/- 0. 12 and 0.26 +/- 0.10, and EDL: 0.95 +/- 0.14 and 0.68 +/- 0.10, for middle-aged and old muscles, respectively). These data support the concept that DHPR-RYR1 uncoupling results in alterations in the voltage-gated sarcoplasmic reticulum Ca2+ release mechanism, decreases in myoplasmic Ca2+ elevation in response to sarcolemmal depolarization, reduced Ca2+ supply to contractile proteins and reduced contraction force with aging.
Asunto(s)
Envejecimiento/fisiología , Canales de Calcio/fisiología , Proteínas Musculares/fisiología , Músculo Esquelético/fisiología , Animales , Calcio/fisiología , Canales de Calcio Tipo L , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344 , Agregación de Receptores , Canal Liberador de Calcio Receptor de RianodinaRESUMEN
The present study investigates the modulation of skeletal muscle L-type Ca2+ channel receptor in response to insulin-like growth factor-1 receptor (IGF-1R) activation. Single extensor digitorum longus and multifiber preparations were isolated from 7- (young), 14- (middle-age) and 28-(old) month- Fisher 344 X Brown Norway rats. Calcium current was potentiated in fibers from young and middle-age rats due to a -13 mV shift in half-activation potential. Fibers from old animals failed to show current potentiation in response to IGF-1R activation. IGF-1 induced a ten-fold increase in the phosphorylation of the L-type Ca2+ channel alpha1 subunit in young and middle-age fibers but failed to induce phosphorylation in old fibers. Addition of 0.5 mM Ca2+ increased the IGF-1 induced phosphorylation in young and middle-age fibers three fold but not in old fibers. The tyrosine kinase inhibitor, genistein, and the PKC inhibitor peptide, 19-36, decreased IGF-1 induced phosphorylation of alpha1 subunit to 15% in young and middle-age fibers but failed to inhibit phosphorylation in old fibers. These results demonstrate that the IGF-1-L-type Ca2+ channel alpha1 subunit signaling is impaired in skeletal muscle fibers from old animals due to alterations in the trk-PKC pathway.
Asunto(s)
Envejecimiento/fisiología , Canales de Calcio/fisiología , Calcio/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Músculo Esquelético/fisiología , Receptor IGF Tipo 1/fisiología , Transducción de Señal , Animales , Canales de Calcio Tipo L , Inhibidores Enzimáticos/farmacología , Genisteína , Técnicas In Vitro , Isoflavonas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Desarrollo de Músculos , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/crecimiento & desarrollo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344RESUMEN
The alpha 7 nicotinic acetylcholine receptor (nAChR) subtype, unlike other neuronal nicotinic receptors, exhibits a relatively high permeability to Ca++ ions. Although Ca++ entry through this receptor subtype has been implicated in various Ca(++)-dependent processes in the central nervous system, little is known about how this receptor modulates mammalian intracellular Ca++ dynamics. Intracellular Ca++ responses evoked by activation of the human alpha 7 nAChRs stably expressed in HEK-293 (human embryonic kidney) cells were studied. Inward current and intracellular Ca++ transients were recorded simultaneously in response to a fast drug application system. Current recordings under whole-cell voltage-clamp and fast ratiometric intracellular Ca++ imaging acquisition were synchronized to drug pulses. The mean peak [Ca++]i observed with 100 microM (-)-nicotine was 356 +/- 48 nM (n = 8). The magnitude of the intracellular Ca++ elevation corresponds to a 20% fractional current carried by Ca++ ions. The EC50 of the intracellular Ca++ responses for (-)-nicotine, (+/-)-epibatidine, 1,1 dimethyl-4-phenyl-piperazinium and acetylcholine were 51, 3.5, 75 and 108 microM, respectively. These EC50 values strongly correlate with those recorded for the cationic inward current through alpha 7 nAChR. alpha-Bungarotoxin, methyllcaconitine or extracellular Ca++ chelation ablated (-)-nicotine-evoked increase in intracellular Ca++ concentration. This study provides evidence that cation influx through the human alpha 7 nAChR is sufficient to mediate a significant, transient, rise in intracellular Ca++ concentration.