RESUMEN
The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.
Asunto(s)
4-Butirolactona/análogos & derivados , Señalización del Calcio , Homoserina/análogos & derivados , Pseudomonas aeruginosa/patogenicidad , 4-Butirolactona/metabolismo , Animales , Apoptosis , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Células Endoteliales/citología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Citometría de Flujo , Homoserina/metabolismo , Humanos , Inflamación/microbiología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Células 3T3 NIH , Pseudomonas aeruginosa/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Virulencia/metabolismoRESUMEN
Mg2+, an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca2+ release channels by competing with Ca2+ at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca2+-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca2+ stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca2+ compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg2+ at the background of 3 mM total ATP in the presence or absence of 1 mM luminal Ca2+. Mg2+ dramatically slowed the rate of activation of RyRs in response to sustained (> or =10-ms) elevations in Ca2+ concentration. Paradoxically, Mg2+ had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (<1-ms) Ca2+ stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca2+ did not affect the peak open probability or the probability of responses to brief Ca2+ signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg2+ is a critical physiological determinant of the dynamic behavior of the RyR channel, which is expected to profoundly influence the fidelity of coupling between L-type Ca2+ channels and RyRs in heart cells.
Asunto(s)
Magnesio/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Calcio/farmacología , Células Cultivadas , Perros , Relación Dosis-Respuesta a DrogaRESUMEN
cADP-Ribose (cADPR) is a novel endogenous messenger that is believed to mobilize Ca(2+) from ryanodine-sensitive Ca(2+) stores. Despite intense research, the precise mechanism of action of cADPR remains uncertain, and experimental findings are contradictory. To elucidate the mechanism of cADPR action, we performed confocal Ca(2+) imaging in saponin-permeabilized rat ventricular myocytes. Exposure of the cells to cADPR resulted in a slow (>2 minutes) and steady increase in the frequency of Ca(2+) sparks. These effects on local release events were accompanied by a significant increase in sarcoplasmic reticulum (SR) Ca(2+) content. In comparison, sensitization of ryanodine receptors (RyRs) by caffeine, a true RyR agonist, caused a rapid (<1 second) and transient potentiation of Ca(2+) sparks followed by a decrease in SR Ca(2+) content. When the increase in the SR load was prevented by partial inhibition of the SR Ca(2+) with thapsigargin, cADPR failed to produce any increase in sparking activity. cADPR had no significant impact on activity of single cardiac RyRs incorporated into lipid bilayers. However, it caused a significant increase in the rate of Ca(2+) uptake by cardiac SR microsomes. Our results suggest that the primary target of cADPR is the SR Ca(2+) uptake mechanism. Potentiation of Ca(2+) release by cADPR is mediated by increased accumulation of Ca(2+) in the SR and subsequent luminal Ca(2+)-dependent activation of RyRs.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Calcio/metabolismo , Ventrículos Cardíacos/metabolismo , Miocardio/metabolismo , Retículo Sarcoplasmático/metabolismo , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/farmacología , Animales , Calcio/farmacocinética , Señalización del Calcio/efectos de los fármacos , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Permeabilidad de la Membrana Celular/efectos de los fármacos , ADP-Ribosa Cíclica , Perros , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Masculino , Microsomas/química , Microsomas/metabolismo , Miocardio/química , Miocardio/citología , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Saponinas/farmacología , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tapsigargina/antagonistas & inhibidores , Tapsigargina/farmacologíaRESUMEN
In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.
Asunto(s)
Calcio/metabolismo , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Cafeína/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Electrofisiología , Fluorescencia , Ventrículos Cardíacos/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Magnesio/farmacología , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tetracaína/farmacología , Tapsigargina/farmacologíaRESUMEN
1. To define the sub-cellular mechanisms of modulation of cardiac excitation-contraction (E-C) coupling by the beta-adrenergic pathway, we carried out confocal Ca(2+) imaging in conjunction with recordings of inward Ca(2+) current in fluo-3-loaded patch-clamped rat ventricular myocytes. 2. Isoproterenol (isoprenaline; ISO) increased the amplitude of the inward Ca(2+) current and the globally measured intracellular Ca(2+) transients. The gain of calcium-induced calcium release (CICR) was increased at all membrane potentials but especially at positive membrane potentials (> +30 mV). ISO dramatically broadened the bell-shaped voltage dependence of intracellular Ca(2+) transients by shifting the descending portion of the curve to very high membrane potentials. 3. The number of local release events (solitary sparks and conglomerates of overlapping sparks) induced by depolarizing steps to +30 mV was increased significantly by ISO. This potentiation of events was due to increased trigger calcium current (I(Ca)) as well the enhanced ability of I(Ca) to induce release. The amplitude and duration of solitary sparks were increased in the presence of ISO. In addition, ISO dramatically increased the proportion and the size ('mass') of clustered events. 4. Exclusion of Na(+) from the intra- and extracellular solutions prevented ISO from enhancing the ability of I(Ca) to trigger sparks. 5. We conclude that beta-adrenergic stimulation enhances the gain of the CICR cascade by increasing the fidelity of dihydropyridine receptor (DHPR)--ryanodine receptor (RyR) coupling and by promoting cross-activation of RyRs in neighbouring release sites. Reverse Na(+)--Ca(2+) exchange (NCX) appears to play a role in the beta-adrenergic enhancement of CICR by effectively contributing to the Ca(2+) trigger.
Asunto(s)
Calcio/fisiología , Miocardio/metabolismo , Receptores Adrenérgicos beta/fisiología , Agonistas Adrenérgicos beta/farmacología , Animales , Conductividad Eléctrica , Electrofisiología , Espacio Extracelular/metabolismo , Ventrículos Cardíacos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Isoproterenol/farmacología , Potenciales de la Membrana/fisiología , Miocardio/citología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/metabolismo , Sodio/fisiologíaRESUMEN
Calcium waves in heart cells are mediated by diffusion-coupled calcium-induced calcium release. The waves propagate in circular fashion. This is counterintuitive in view of the accepted ultrastructure of the cardiac myocyte. The density of calcium release sites in the transverse direction is four times higher than in the longitudinal direction. Simulations with release sites localized along Z-lines and isotropic diffusion yielded highly elliptical, nonphysiological waves. We hypothesized that subcellular organelles counteracted the higher release site density along the Z-lines by acting as transverse diffusion barriers and sites of active calcium uptake. We quantified the reduction of transverse diffusion by microinjecting cells with the nonreactive dye fluorescein. The ratio of the radial diffusion coefficient to the longitudinal coefficient was 0.39. Inhibition of mitochondrial uptake by rotenone accelerated the wave in the transverse direction. Simulations with release sites clustered at the Z-lines and a transverse diffusion coefficient 50% of the longitudinal coefficient generated waves of ellipticity 2/1 (major axis along the Z-line). Introducing additional release sites between the Z-lines at a density 20% of that on the Z-lines produced circular waves. The experiments and simulations support the presence of transverse diffusion barriers, additional uptake sites, and possibly intermediate release sites as well.
Asunto(s)
Señalización del Calcio/fisiología , Miocardio/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Difusión , Fluoresceína , Polarización de Fluorescencia , Técnicas In Vitro , Cinética , Microscopía Confocal , Modelos Cardiovasculares , Miocardio/citología , Ratas , Ratas Sprague-Dawley , Sarcómeros/metabolismoRESUMEN
We have compared the effects of the sarcoplasmic reticulum (SR) Ca(2+) release inhibitor, ruthenium red (RR), on single ryanodine receptor (RyR) channels in lipid bilayers, and on Ca(2+) sparks in permeabilized rat ventricular myocytes. Ruthenium red at 5 microM inhibited the open probability (P(o)) of RyRs approximately 20-50-fold, without significantly affecting the conductance or mean open time of the channel. At the same concentration, RR inhibited the frequency of Ca(2+) sparks in permeabilized myocytes by approximately 10-fold, and reduced the amplitude of large amplitude events (with most probable localization on the line scan) by approximately 3-fold. According to our theoretical simulations, performed with a numerical model of Ca(2+) spark formation, this reduction in Ca(2+) spark amplitude corresponds to an approximately 4-fold decrease in Ca(2+) release flux underlying Ca(2+) sparks. Ruthenium red (5 microM) increased the SR Ca(2+) content by approximately 2-fold (from 151 to 312 micromol/l cytosol). Considering the degree of inhibition of local Ca(2+) release events, the increase in SR Ca(2+) load by RR, and the lack of effects of RR on single RyR open time and conductance, we have estimated that Ca(2+) sparks under normal conditions are generated by openings of at least 10 single RyRs.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Corazón/fisiología , Miocardio/citología , Rojo de Rutenio/farmacología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Cafeína/farmacología , Señalización del Calcio/efectos de los fármacos , Permeabilidad de la Membrana Celular , Femenino , Corazón/efectos de los fármacos , Ventrículos Cardíacos , Cinética , Masculino , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacosRESUMEN
The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.
Asunto(s)
Señalización del Calcio/fisiología , Corazón/fisiología , Miocardio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Acetatos/farmacología , Algoritmos , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Quelantes/farmacología , Perros , Etilenodiaminas/farmacología , Corazón/efectos de los fármacos , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Cinética , Membrana Dobles de Lípidos , Microsomas/metabolismo , Modelos Biológicos , Técnicas de Placa-Clamp , Fotólisis , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
1. We carried out confocal Ca2+ imaging in myocytes permeabilized with saponin in 'internal' solutions containing: MgATP, EGTA and fluo-3 potassium salt. 2. Permeabilized myocytes exhibited spontaneous Ca2+ sparks and waves similar to those observed in intact myocytes loaded with fluo-3 AM. 3. In the presence of 'low' [EGTA] (0.05 mM), Ca2+ waves arose regularly, even at relatively low [Ca2+] (50-100 nM, free). Increasing [EGTA] resulted in decreased frequency and propagation velocity of Ca2+ waves. Propagating waves were completely abolished at [EGTA] > 0.3 mM. 4. The frequency of sparks increased as a function of [Ca2+] (50-400 nM range) with no sign of a high affinity Ca2+-dependent inactivation process. 5. The rate of occurrence of Ca2+ sparks was increased by calmodulin and cyclic adenosine diphosphate-ribose (cADPR).
Asunto(s)
Señalización del Calcio/fisiología , Calcio/fisiología , Miocardio/metabolismo , Saponinas/farmacología , Adenosina Difosfato Ribosa/farmacología , Animales , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Calmodulina/fisiología , Permeabilidad de la Membrana Celular/fisiología , Separación Celular , Citosol/metabolismo , Ácido Egtácico/farmacología , Espacio Extracelular/metabolismo , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Microscopía Confocal , Miocardio/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Channel adaptation is a basic property of the sarcoplasmic reticulum Ca2+-release channels/ryanodine receptors (RyRs). It allows channel activity to decay during sustained increases in the concentration of activating Ca2+. Despite the potential physiological importance of this self-confining process, its molecular mechanism is not well understood. To define the mechanism of adaptation we studied the dynamics of cardiac Ca2+-release channel (RyR) gating using the planar lipid bilayer technique in combination with photolysis of caged Ca2+ (DM-nitrophen). Channels activated by rapid and sustained increases in Ca2+ concentration (from 0.1 to 0.5 micromol/l) displayed three distinct gating modes, manifested as current records with frequent and long openings (H-mode), with rare and short openings (L-mode), and with no openings (I-mode). H-mode channel activity occurred primarily at early times while L- and I-modes predominated at late times after the rapid Ca2+ concentration increase. The decrease in probability of H-mode, mirrored by an increase in the probability of the I-mode, proceeded with a time constant similar to that observed for spontaneous decay in channel activity (i.e., adaptation) in ensemble average records. These results indicate that RyR adaptation transpires by a shift of channel gating from a high open probability mode to low open probability and inactivated modes of the channel.
Asunto(s)
Calcio/metabolismo , Activación del Canal Iónico/fisiología , Miocardio/química , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Perros , Cinética , Membrana Dobles de Lípidos/metabolismo , Microsomas/química , Miocardio/metabolismo , Miocardio/ultraestructura , Fotólisis , Retículo Sarcoplasmático/ultraestructuraRESUMEN
1. We used confocal Ca2+ imaging and fluo-3 to investigate the transition of localized Ca2+ releases induced by focal caffeine stimulation into propagating Ca2+ waves in isolated rat ventricular myocytes. 2. Self-sustaining Ca2+ waves could be initiated when the cellular Ca2+ load was increased by elevating the extracellular [Ca2+] ([Ca2+]o) and they could also be initiated at normal Ca2+ loads when the sensitivity of the release sites to cytosolic Ca2+ was enhanced by low doses of caffeine. When we prevented the accumulation of extra Ca2+ in the luminal compartment of the sarcoplasmic reticulum (SR) with thapsigargin, focal caffeine pulses failed to trigger self-sustaining Ca2+ waves on elevation of [Ca2+]o. Inhibition of SR Ca2+ uptake by thapsigargin in cells already preloaded with Ca2+ above normal levels did not prevent local Ca2+ elevations from triggering propagating waves. Moreover, wave velocity increased by 20 %. Tetracaine (0.75 mM) caused transient complete inhibition of both local and propagating Ca2+ signals, followed by full recovery of the responses due to increased SR Ca2+ accumulation. 3. Computer simulations using a numerical model with spatially distinct Ca2+ release sites suggested that increased amounts of releasable Ca2+ might not be sufficient to generate self-sustaining Ca2+ waves under conditions of Ca2+ overload unless the threshold of release site Ca2+ activation was set at relatively low levels (< 1.5 microM). 4. We conclude that the potentiation of SR Ca2+ release channels by luminal Ca2+ is an important factor in Ca2+ wave generation. Wave propagation does not require the translocation of Ca2+ from the spreading wave front into the SR. Instead, it relies on luminal Ca2+ sensitizing Ca2+ release channels to cytosolic Ca2+.
Asunto(s)
Calcio/fisiología , Corazón/fisiología , Algoritmos , Anestésicos Locales/farmacología , Compuestos de Anilina , Animales , Cafeína/farmacología , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Estimulantes del Sistema Nervioso Central/farmacología , Simulación por Computador , Citosol/efectos de los fármacos , Citosol/metabolismo , Electrofisiología , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes , Corazón/efectos de los fármacos , Membrana Dobles de Lípidos , Microscopía Confocal , Modelos Biológicos , Miocardio/citología , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Tapsigargina/farmacología , XantenosRESUMEN
The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca2+ (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca2+ on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low trans Ca2+ (20 microM), increasing cis Ca2+ from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating cis Ca2+ above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca2+] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca2+ enhanced the sensitivity of the channel to activating cytosolic Ca2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca2+. Potentiation of Po by increased luminal Ca2+ occurred irrespective of whether the electrochemical gradient for Ca2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca2+-sensitive site(s) at the luminal face of the channel or associated protein.
Asunto(s)
Calcio/metabolismo , Miocardio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Calcio/farmacología , Citosol/metabolismo , Perros , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Potenciales de la Membrana , Microsomas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
1. Confocal Ca2+ imaging was used to measure spontaneous release events (Ca2+ sparks) in fluo-3-loaded isolated rat ventricular myocytes. 2. The microscopic Ca2+ release flux underlying Ca2+ sparks was derived by adapting the methods used previously to describe macroscopic Ca2+ release from cell-averaged Ca2+ transients. 3. The magnitude of the local release fluxes varied from 2 to 5 microM ms-1, depending on SR Ca2+ loading conditions. Following spontaneous activation, the release flux rapidly decayed (tau = 6-12 ms). The rate of termination of release flux was found to be directly related to the magnitude of the flux (r2 = 0.88). 4. The rate of termination of local release flux was slowed in the presence of FK506, a compound that is known to reduce inactivation of SR Ca2+ channels in vitro. 5. These results suggest that termination of release flux during sparks is not due to a spontaneous stochastic decay process or local depletion of Ca2+ from the SR, but rather involves an active extinguishing mechanism such as Ca2+-dependent inactivation or adaptation.
Asunto(s)
Calcio/metabolismo , Corazón/fisiología , Retículo Sarcoplasmático/metabolismo , Algoritmos , Compuestos de Anilina , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Células Cultivadas , Colorantes Fluorescentes , Ventrículos Cardíacos , Cinética , Microscopía Confocal , Modelos Químicos , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Retículo Sarcoplasmático/efectos de los fármacos , Tacrolimus/farmacología , XantenosRESUMEN
1. Confocal microfluorometry was used to study the effects of tetracaine on spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) in isolated rat ventricular myocytes. 2. At low concentrations (0.25-1.25 mM), tetracaine caused an initial inhibition of spontaneous release events (Ca2+ sparks) and Ca2+ waves, which was followed by a gradual increase in Ca2+ release activity. The frequency and magnitude of sparks were first decreased and then increased with respect to control levels. At high concentrations (> 1.25 mM), tetracaine abolished all forms of spontaneous release. 3. Exposure of the myocytes to tetracaine resulted in a gradual increase in the SR Ca2+ load as indexed by changes in the magnitude of caffeine-induced Ca2+ transients. 4. In cardiac SR Ca(2+)-release channels incorporated into lipid bilayers, tetracaine (> 0.25 mM) induced a steady inhibition of channel activity. Addition of millimolar Ca2+ to the luminal side of the channel caused an increase in channel open probability under control conditions as well as in the presence of various concentrations of tetracaine. 5. We conclude that the primary effect of tetracaine on SR Ca(2+)-release channels is inhibition of channel activity both in vitro and in situ. The ability of tetracaine to reduce spark magnitude suggests that these events are not due to activation of single channels or non-reducible clusters of channels and, therefore, supports the multichannel origin of sparks. We propose that the paradoxical late potentiation of release by submaximal concentrations of tetracaine is caused by a gradual increase in SR Ca2+ load and subsequent activation of the Ca(2+)-release channels by Ca2+ inside the SR.
Asunto(s)
Anestésicos Locales/farmacología , Calcio/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Miocardio/citología , Tetracaína/farmacología , Animales , Cafeína/farmacología , Calcio/farmacocinética , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , Electrofisiología , Ventrículos Cardíacos/química , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Microscopía Confocal , Microsomas/química , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/ultraestructura , Miocardio/química , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Sarcolema/química , Sarcolema/efectos de los fármacos , Sarcolema/metabolismoRESUMEN
Single cardiac ryanodine receptor (RyR) channel adaptation was previously defined with Ca2+ stimuli produced by flash photolysis of DM-nitrophen (caged-Ca+2). Photolysis of DM-nitrophen induced a very fast Ca+2 overshoot (Ca+2 spike) at the leading edge of the Ca+2 stimuli. It has been suggested that adaptation (tau approximately 1.3 s) may reflect Ca+2 slowly coming off the RyR Ca+2 activation sites following the faster Ca+2 spike (tau approximately 1 ms). This concern was addressed by defining the Ca2+ deactivation kinetics of single RyR channels in response to a rapid reduction in free Ca2+ concentration ([Ca2+]FREE). The [Ca2+]FREE was lowered by photolysis of Diazo-2. Single RyR channels deactivated (tau approximately 5.3 ms) quickly in response to the photolytically induced [Ca2+]FREE reduction. Improved estimates of the Ca2+ spike time course indicate that the Ca2+ spike is considerably faster (10-100-fold) than previously thought. Our data suggest that single RyRs are not significantly activated by fast Ca2+ spikes and that RyR adaptation is not due to deactivation following the fast Ca2+ spike. Thus, RyR adaptation may have an important impact on Ca2+ signaling in heart.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Retículo Sarcoplasmático/metabolismo , Acetatos , Animales , Calcio/farmacología , Quelantes , Compuestos de Diazonio , Perros , Etilenodiaminas , Técnicas In Vitro , Cinética , Membrana Dobles de Lípidos/metabolismo , Técnicas de Placa-Clamp , Fenoxiacetatos , Fosfolípidos/metabolismo , Fotólisis , Canal Liberador de Calcio Receptor de Rianodina , Transducción de SeñalRESUMEN
To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca2+ on the Ca2+ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca2+ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca2+ overload, induced by elevation of extracellular [Ca2+], Ca2+ sparks represented single populations of events. During Ca2+ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 microm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca2+ sparks, however, was not altered. Changes in the properties of Ca2+ sparks were accompanied by only an approximately 30% increase in the SR Ca2+ content, as determined by emptying the intracellular Ca2+ stores using caffeine. When single Ca2+ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca2+ (approximately 100 nM) and ATP (3 mM), elevation of Ca2+ on the luminal side from 20 microM to 0.2-20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (Po). This potentiation of Po was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca2+ was greater at low levels of cytoplasmic [Ca2+] than at high levels of cytoplasmic [Ca2+], and no effect of luminal Ca2+ was observed to occur in channels activated by 0.5-50 microM cytoplasmic Ca2+ in the absence of ATP. Our results suggest that SR Ca2+ release channels are modulated by SR intraluminal Ca2+. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca2+ release in cardiac myocytes
Asunto(s)
Calcio/metabolismo , Calcio/farmacología , Miocardio/metabolismo , Retículo Sarcoplasmático/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Cesio/metabolismo , Perros , Ventrículos Cardíacos/ultraestructura , Microscopía Confocal , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/fisiología , Miocardio/ultraestructura , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/efectos de los fármacosRESUMEN
Laser scanning confocal imaging was used to monitor release of Ca2+ from localized regions in a skeletal muscle cell line with sparsely distributed Ca2+ release sites. The goal was to distinguish between two schemes proposed to explain the phenomenon of "quantal" Ca2+ release from caffeine-sensitive Ca2+ stores in muscle and other tissues: (1) all-or-none (true quantal) Ca2+ release from functionally discrete stores that have different sensitivities to caffeine; or (2) adaptive behavior of individual release sites, each responding transiently and repeatedly to incremental caffeine doses. Our results showed that Ca2+ release induced by K+ or caffeine occurs in discrete loci within the cell. The image areas and fluorescence intensities of some of these evoked local signals were similar to those of Ca2+ sparks that were observed under resting conditions and which are believed to be due to spontaneous activation of single release units. In contrast to the expectations imposed by quantal models, incremental doses of caffeine activated the same sets of release sites throughout the cell. Ca2+ release, at a given site, triggered by a submaximal dose of caffeine was transient and could be reactivated by addition of a higher caffeine dose, showing the same type of adaptive behavior as measured globally from larger areas of the cell. These results suggest that incremental Ca2+ release is accounted for by adaptive behavior of individual Ca2+ release sites.
Asunto(s)
Calcio/metabolismo , Músculo Esquelético/metabolismo , Adaptación Fisiológica , Animales , Sitios de Unión , Cafeína/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Línea Celular , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Ratones , Microscopía Confocal , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/ultraestructura , Potasio/farmacología , Canal Liberador de Calcio Receptor de RianodinaRESUMEN
Ryanodine receptors have recently been shown to undergo an unusual kind of inactivation process termed adaptation, which bears similarities to the transient calcium releases induced in other systems by successive incremental additions of inositol-1,4,5-trisphosphate. Such releases are sometimes termed "quantal". In this study we report that many agonists induce similar behavior in muscle sarcoplasmic reticulum and that the responses depend not on the calcium pumps therein but rather on the ryanodine receptors. The chemical diversity of these agonists makes it very unlikely that adaptation simply affects the sensitivity of the receptor to agonists at any one binding site. More likely, this result indicates that adaptive behavior of ryanodine receptors results whenever the ryanodine receptor is activated and that this process affects the action of most, if not all, agonists. Evidence is presented suggesting that the releases observed do not represent all-or-none releases from vesicle subpopulations (true quantal behavior) but rather seem to involve partial release from more homogeneously sensitive stores, a process referred to here as adaptation or increment detection.