RESUMO
To study Ca(2+) fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in approximately 20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 +/- 5 microm, indicating that Ca(2+) transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca(2+)](ER) averaged 371 +/- 21 microm at rest and decreased to 133 +/- 14 microm and 59 +/- 5 microm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca(2+) uptake was prevented by oligomycin and rotenone or when Ca(2+) efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca(2+) taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca(2+) than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca(2+) uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca(2+) release and recycle a substantial portion of the captured Ca(2+) back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca(2+) signals and the filling state of neighboring ER regions.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Linhagem Celular , Clonazepam/análogos & derivados , Clonazepam/farmacologia , Citosol/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Corantes Fluorescentes , Células HeLa , Histamina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oligomicinas/farmacologia , Rotenona/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Tapsigargina/farmacologia , Tiazepinas/farmacologiaRESUMO
We recorded Ca2+ sparks and spontaneous transient outward currents (STOCs) simultaneously in smooth muscle cells using whole-cell patch recording and a unique, high-speed widefield digital imaging system to monitor fluo-3 fluorescence in both two and three dimensions (2D and 3D). In 2D imaging, the correlation between the amplitude of a spark and its corresponding STOC was a weak one, and 27 % of the sparks failed to cause STOCs. The STOCless sparks were not significantly different in amplitude from those that caused STOCs. Three-dimensional imaging disclosed that STOCless sparks were located close to the cell surface, and on average their apparent distance from the cell surface was not significantly different from the sparks that cause STOCs. Statistical evaluation of spark clusters disclosed that there were regions of the cell where the probability of spark occurrence was high and others where it was quite low.
Assuntos
Cálcio/fisiologia , Esôfago/fisiologia , Músculo Liso/fisiologia , Compostos de Anilina , Animais , Gatos , Membrana Celular/fisiologia , Condutividade Elétrica , Esôfago/citologia , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Músculo Liso/citologia , Técnicas de Patch-Clamp , XantenosRESUMO
Ca(2+) sparks are highly localized cytosolic Ca(2+) transients caused by a release of Ca(2+) from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca(2+) in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca(2+) indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca(2+) released into the cytosol, and its rate of rise is proportional to the Ca(2+) current flowing through the RyRs during a spark (I(Ca(spark))). Thus, Ca(2+) currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of I(Ca(spark)) in different sparks varies more than fivefold, Ca(2+) sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca(2+) current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca(2+)] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca(2+) concentration resulting from the measured range of I(Ca(spark)). At the onset of a spark, the Ca(2+) concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca(2+)](EC50) for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca(2+)] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca(2+) action on a variety of molecular targets within cellular microdomains.
Assuntos
Sinalização do Cálcio , Cálcio/fisiologia , Membranas Intracelulares/fisiologia , Canais de Potássio/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Bradicinina/metabolismo , Bufo marinus , Citosol/metabolismo , Condutividade Elétrica , Fluorescência , Processamento de Imagem Assistida por Computador , Canais Iônicos/metabolismo , Cinética , Microscopia , Modelos Biológicos , Técnicas de Patch-ClampRESUMO
1. The Ca(2+)-sensitive fluorescent indicator rhod-2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura-2 was used in combination with rhod-2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. 2. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half-time (t1/2) to peak for the increase in [Ca2+]m was considerably longer than the t1/2 to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. 3. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t1/2 to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. 4. Using a wide-field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. 5. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. 6. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR.
Assuntos
Cálcio/metabolismo , Homeostase/fisiologia , Mitocôndrias/metabolismo , Músculo Liso/metabolismo , Animais , Bufo marinus , Cafeína/farmacologia , Células Cultivadas , Citoplasma/metabolismo , Estimulação Elétrica , Corantes Fluorescentes/farmacocinética , Compostos Heterocíclicos com 3 Anéis , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Microscopia de Vídeo , Músculo Liso/citologia , Técnicas de Patch-Clamp , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , EstômagoRESUMO
Localized, transient elevations in cytosolic Ca2+, known as Ca2+ sparks, caused by Ca2+ release from sarcoplasmic reticulum, are thought to trigger the opening of large conductance Ca2+-activated potassium channels in the plasma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca2+ concentration within the sarcoplasmic reticulum and a Ca2+ spark and that between a Ca2+ spark and a STOC are not well defined or fully understood. To address these problems, we have employed two approaches using single patch-clamped smooth muscle cells freshly dissociated from toad stomach: a high speed, wide-field imaging system to simultaneously record Ca2+ sparks and STOCs, and a method to simultaneously measure free global Ca2+ concentration in the sarcoplasmic reticulum ([Ca2+]SR) and in the cytosol ([Ca2+]CYTO) along with STOCs. At a holding potential of 0 mV, cells displayed Ca2+ sparks and STOCs. Ca2+ sparks were associated with STOCs; the onset of the sparks coincided with the upstroke of STOCs, and both had approximately the same decay time. The mean increase in [Ca2+]CYTO at the time and location of the spark peak was approximately 100 nM above a resting concentration of approximately 100 nM. The frequency and amplitude of spontaneous Ca2+ sparks recorded at -80 mV were unchanged for a period of 10 min after removal of extracellular Ca2+ (nominally Ca2+-free solution with 50 microM EGTA), indicating that Ca2+ influx is not necessary for Ca2+sparks. A brief pulse of caffeine (20 mM) elicited a rapid decrease in [Ca2+]SR in association with a surge in [Ca2+]CYTO and a fusion of STOCs, followed by a fast restoration of [Ca2+]CYTO and a gradual recovery of [Ca2+]SR and STOCs. The return of global [Ca2+]CYTO to rest was an order of magnitude faster than the refilling of the sarcoplasmic reticulum with Ca2+. After the global [Ca2+]CYTO was fully restored, recovery of STOC frequency and amplitude were correlated with the level of [Ca2+]SR, even though the time for refilling varied greatly. STOC frequency did not recover substantially until the [Ca2+]SR was restored to 60% or more of resting levels. At [Ca2+]SR levels above 80% of rest, there was a steep relationship between [Ca2+]SR and STOC frequency. In contrast, the relationship between [Ca2+]SR and STOC amplitude was linear. The relationship between [Ca2+]SR and the frequency and amplitude was the same for Ca2+ sparks as it was for STOCs. The results of this study suggest that the regulation of [Ca2+]SR might provide one mechanism whereby agents could govern Ca2+ sparks and STOCs. The relationship between Ca2+ sparks and STOCs also implies a close association between a sarcoplasmic reticulum Ca2+ release site and the Ca2+-activated potassium channels responsible for a STOC.
Assuntos
Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Bufo marinus , Citosol/metabolismo , Estimulação Elétrica , Eletrofisiologia , Espaço Extracelular/metabolismo , Fura-2 , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , Indicadores e Reagentes , Potenciais da Membrana/fisiologia , Microscopia Confocal , Músculo Liso/citologia , Músculo Liso/ultraestrutura , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Retículo Sarcoplasmático/ultraestruturaRESUMO
1. Local changes in cytosolic [Ca2+] were imaged with a wide-field, high-speed, digital imaging system while membrane currents were simultaneously recorded using whole-cell, perforated patch recording in freshly dissociated guinea-pig tracheal myocytes. 2. Depending on membrane potential, Ca2+ sparks triggered 'spontaneous' transient inward currents (STICs), 'spontaneous' transient outward currents (STOCs) and biphasic currents in which the outward phase always preceded the inward (STOICs). The outward currents resulted from the opening of large-conductance Ca2+-activated K+ (BK) channels and the inward currents from Ca2+-activated Cl- (ClCa) channels. 3. A single Ca2+ spark elicited both phases of a STOIC, and sparks originating from the same site triggered STOCs, STICs and STOICs, depending on membrane potential. 4. STOCs had a shorter time to peak (TTP) than Ca2+ sparks and a much shorter half-time of decay. In contrast, STICs had a somewhat longer TTP than sparks but the same half-time of decay. Thus, the STIC, not the STOC, more closely reflected the time course of cytosolic Ca2+ elevation during a Ca2+ spark. 5. These findings suggest that ClCa channels and BK channels may be organized spatially in quite different ways in relation to points of Ca2+ release from intracellular Ca2+ stores. The results also suggest that Ca2+ sparks may have functions in smooth muscle not previously suggested, such as a stabilizing effect on membrane potential and hence on the contractile state of the cell, or as activators of voltage-gated Ca2+ channels due to depolarization mediated by STICs.
Assuntos
Sinalização do Cálcio/fisiologia , Canais de Cloreto/metabolismo , Canais de Potássio/metabolismo , Traqueia/metabolismo , Algoritmos , Animais , Bradicinina/metabolismo , Estimulação Elétrica , Eletrofisiologia , Cobaias , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Fatores de Tempo , Traqueia/citologiaRESUMO
Ca2+ currents (ICa) and cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in isolated gastric myocytes from Bufo marinus using whole cell voltage clamp and fura 2, respectively. After a conditioning train of depolarizing pulses, high-voltage-activated ICa (test potential of +10 mV) was increased, returning to control values after approximately 85 s. This enhancement was [Ca2+]c dependent, with a maximal increase at approximately 600 nM [Ca2+]c. During the conditioning train, ICa measured at 70 ms, which provides a measure of high-voltage-activated current, initially decreased with each successive pulse to a minimum of 56 +/- 5% of the first pulse in the train. Thereafter, the 70-ms current showed considerable recovery. Blockade of calmodulin activity with a peptide (RS20) or calmidazolium did not affect the early inhibition but did abolish current recovery. A peptide inhibitor of calmodulin-dependent protein kinase II (CK3AA) had similar effects. Substraction of currents measured in the presence and absence of RS20 revealed a 2-s delay between the start of the train and the onset of current enhancement. It was also observed that low-voltage-activated current (test potential of -17 mV) was reduced to 76 +/- 7% of control 5 s after the conditioning train; this inhibition recovered to 92 +/- 4% after 35 s and was not dependent on [Ca2+]c elevation.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/farmacologia , Músculo Liso/fisiologia , Estômago/fisiologia , Sequência de Aminoácidos , Animais , Bário/farmacologia , Bufo marinus , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Calmodulina/antagonistas & inibidores , Proteínas de Ligação a Calmodulina/farmacologia , Células Cultivadas , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Fura-2 , Imidazóis/farmacologia , Cinética , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Espectrometria de FluorescênciaRESUMO
The purinergic rP2X7 receptor expressed in a number of heterologous systems not only functions as a cation channel but also gives rise to a P2Z-like response, i.e. a reversible membrane permeabilization that allows the passage of molecules with molecular masses of > or = 300 Da. We investigated the properties of rP2X7 receptors expressed in Xenopus oocytes. In two-electrode voltage-clamp experiments, ATP or BzATP caused inward currents that were abolished or greatly diminished when NMDG+ or choline replaced Na+ as the principal external cation. In fluorescent dye experiments, BzATP application did not result in entry of the fluorophore YO-PRO-1(2+). Thus, rP2X7 expression in Xenopus oocytes does not by itself give rise to the pore-forming P2Z phenotype, suggesting that ancillary factors are involved.
Assuntos
Permeabilidade da Membrana Celular , Canais Iônicos/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Benzoxazóis , Eletrofisiologia , Corantes Fluorescentes/metabolismo , Expressão Gênica , Meglumina/metabolismo , Microinjeções , Oócitos , Técnicas de Patch-Clamp , Fenótipo , Compostos de Quinolínio , RNA Complementar/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Proteínas Recombinantes/metabolismo , XenopusRESUMO
1. Whole-cell and single-channel currents elicited by extracellular ATP were studied in freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus using standard patch clamp and microfluorimetric techniques. 2. This ATP-gated cation channel shares a number of pharmacological and functional properties with native rat myometrium receptors, certain native P2Z purinoceptors and the recently cloned P2X7 purinoceptor. But, unlike the last two, the ATP-gated channel does not mediate the formation of large non-specific pores. Thus, it may represent a novel member of the P2X or P2Z class. 3. Extracellular application of ATP (> or = 150 microM) elicited an inward whole-cell current at negative holding potentials that was inwardly rectifying and showed no sign of desensitization. Na+, Cs+ and, to a lesser degree, the organic cation choline served as charge carriers, but Cl- did not. Ratiometric fura-2 measurements indicated that the current is carried in part by Ca2+. The EC50 for ATP was 700 microM in solutions with a low divalent cation concentration. 4. ATP (> or = 100 microM) at the extracellular surface of cell-attached or excised patches elicited inwardly rectifying single-channel currents with a 22 pS conductance. Cl- did not serve as a charge carrier but both Na+ and Cs+ did, as did choline to a lesser extent. The mean open time of the channel was quite long, with a range in hundreds of milliseconds at a holding potential of -70 mV. 5. Mg2+ and Ca2+ decreased the magnitude of the ATP-induced whole-cell currents. Mg2+ decreased both the amplitude and the activity of ATP-activated single-channel currents. 6. ADP, UTP, P1, P5-di-adenosine pentaphosphate (AP5A), adenosine and alpha, beta-methylene ATP (alpha, beta-Me-ATP) did not induce significant whole-cell current. ATP-gamma-S and 2-methylthio ATP (2-Me-S-ATP) were significantly less effective than ATP in inducing whole-cell currents, whereas benzoylbenzoyl ATP (BzATP) was more effective. BzATP, alpha, beta-Me-ATP, ATP-gamma-S and 2-Me-S-ATP induced single-channel currents, but a higher concentration of alpha, beta-Me-ATP was required. 7. BzATP did not induce the formation of large non-specific pores, as assayed using mag-fura-2 as a high molecular mass probe.
Assuntos
Trifosfato de Adenosina/fisiologia , Ativação do Canal Iônico/fisiologia , Músculo Liso/fisiologia , Receptores Purinérgicos P2/fisiologia , Estômago/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Bufo marinus , Cálcio/metabolismo , Cátions Bivalentes/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Estimulação Elétrica , Eletrofisiologia , Proteínas de Ligação ao GTP/metabolismo , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Receptores Purinérgicos P2/efeitos dos fármacos , Estômago/efeitos dos fármacosRESUMO
1. using standard single channel patch clamp techniques we studied the stretch sensitivity of a 20 pS K(+)-selective channel which is activated by fatty acids and found in freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus. 2. A pulse of suction applied to the back of the patch pipette in order to stretch the membrane resulted in activation of this K+ channel. A train of suction pulses resulted in a gradually increased level of channel activity during each successive pulse, as well as an increase in baseline activity between pulses. This pattern contrasts markedly with many other stretch-activated channels whose activation is limited to the duration of the suction pulse. 3. Application of fatty acids augmented the response to stretch. In contrast, application of 10 microM defatted albumin, which removes fatty acids from membranes, rapidly and reversibly decreased the response to stretch. 4. These results are consistent with the hypothesis that fatty acids which are generated by mechanical stimuli, perhaps by mechanically activated phospholipases, are the intermediaries in activation of certain mechanically sensitive ion channels.
Assuntos
Ácidos Graxos/farmacologia , Músculo Liso/fisiologia , Canais de Potássio/fisiologia , Albuminas/farmacologia , Animais , Bufo marinus , Potenciais da Membrana/fisiologia , Ácido Mirístico , Ácidos Mirísticos/farmacologia , Técnicas de Patch-Clamp , Estimulação Física , Canais de Potássio/efeitos dos fármacos , Fatores de TempoRESUMO
A variety of fatty acids increase the activity of certain types of K+ channels. This effect is not dependent on the three enzymatic pathways that convert arachidonic acid to various bioactive oxygenated metabolites. One type of K+ channel in toad stomach smooth muscle cell membranes in activated by fatty acids and other single chain lipids which possess both a negatively charged head group and a sufficiently hydrophobic acyl chain. Neutral lipids have no effect on K+ channel activity, while positively charged lipids with a sufficiently hydrophobic acyl chain suppress channel activity. Acyl Coenzyme A's, which do not flip across the bilayer, act only from the cytosolic surface of the membrane, suggesting that the binding site for channel activation is also located there. This fatty acid-activated channel is also activated by membrane stretch. Moreover, this mechanical response is either mediated or modulated by fatty acids. Thus, fatty acids and other charged single chain lipids may comprise another class of first or second messenger molecules that target ion channels.
Assuntos
Acil Coenzima A/farmacologia , Ácidos Graxos/farmacologia , Músculo Liso/metabolismo , Canais de Potássio/fisiologia , Acil Coenzima A/química , Animais , Bufo marinus , Técnicas In Vitro , Modelos Biológicos , Técnicas de Patch-Clamp , Estimulação Física , Estimulação Química , Estômago/citologiaRESUMO
We determined the structural features necessary for fatty acids to exert their action on K+ channels of gastric smooth muscle cells. Examination of the effects of a variety of synthetic and naturally occurring lipid compounds on K+ channel activity in cell-attached and excised membrane patches revealed that negatively charged analogs of medium to long chain fatty acids (but not short chain analogs) as well as certain other negatively charged lipids activate the channels. In contrast, positively charged, medium to long chain analogs suppress activity, and neutral analogs are without effect. The key requirements for effective compounds seem to be a sufficiently hydrophobic domain and the presence of a charged group. Furthermore, those negatively charged compounds unable to "flip" across the bilayer are effective only when applied at the cytosolic surface of the membrane, suggesting that the site of fatty acid action is also located there. Finally, because some of the effective compounds, for example, the fatty acids themselves, lysophosphatidate, acyl Coenzyme A, and sphingosine, are naturally occurring substances and can be liberated by agonist-activated or metabolic enzymes, they may act as second messengers targeting ion channels.
Assuntos
Lipídeos/química , Lipídeos/fisiologia , Músculo Liso/metabolismo , Canais de Potássio/fisiologia , Acil Coenzima A/farmacologia , Aminas/química , Aminas/farmacologia , Animais , Bufo marinus , Separação Celular , Eletroquímica , Eletrofisiologia , Ácidos Graxos/farmacologia , Bicamadas Lipídicas , Lisofosfolipídeos/química , Lisofosfolipídeos/farmacologia , Músculo Liso/citologia , Esfingosina/farmacologiaRESUMO
The sodium/calcium (Na+/Ca2+) exchanger is often considered to be a key regulator of the cytoplasmic calcium concentration ([Ca2+]) in smooth muscle but neither its precise role in Ca2+ homeostasis nor even its existence in smooth muscle are generally agreed upon. Here we directly assessed the role Na+/Ca2+ exchange plays in regulating [Ca2+] in single voltage-clamped smooth muscle cells. Following an elevation of [Ca2+], its decline was found to have both voltage-dependent and voltage-independent components. The voltage-dependent component was abolished when Na+ was removed from the external bathing solution. During the fall of [Ca2+] a small and declining Na(+)-dependent inward current was observed of a magnitude predicted by 3:1 Na+/Ca2+ exchange stoichiometry. At [Ca2+] above 400 nM the principal efflux of Ca2+ above rest was attributed to this Na(+)-dependent removal mechanism. These results establish that a Na+/Ca2+ exchanger exists in smooth muscle and argue that it can regulate [Ca2+] at physiological Ca2+ concentrations.
Assuntos
Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Músculo Liso/metabolismo , Sódio/metabolismo , Animais , Bufo marinus , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Citoplasma/fisiologia , Eletrofisiologia , Técnicas In Vitro , Cinética , Músculo Liso/fisiologia , Trocador de Sódio e CálcioRESUMO
Large-conductance, Ca(2+)-activated K+ channels were identified in single smooth muscle cells freshly isolated from rabbit superior mesenteric artery. They typically showed a reversal potential close to 0 mV in excised, inside-out patches in symmetric 130 mmol/L [K+] with a unitary conductance of 260 pS, and increased activity at more positive potentials and/or when [Ca2+] was raised at the cytosolic surface of the membrane. Both in cell-attached and in excised, inside-out configurations, stretching the membrane patch by applying suction to the back of the patch pipette increased the activity of these channels without changing either the unitary conductance or the voltage sensitivity of the channel. Stretch activation was repeatedly seen in inside-out patches when both surfaces were bathed with a 0 Ca2+ solution containing 2 or 5 mmol/L EGTA to chelate trace amounts of Ca2+, making it highly improbable that stretch activation could be secondary to a stretch-induced flux of Ca2+. Consequently, stretch activation of large-conductance, Ca(2+)-activated K+ channels in mesenteric artery smooth muscle cells seems to be due to a direct effect of stretch on the channel itself or on some closely associated, membrane-bound entity.
Assuntos
Cálcio/fisiologia , Músculo Liso Vascular/metabolismo , Canais de Potássio/metabolismo , Animais , Membrana Celular/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Retroalimentação/fisiologia , Hipertensão/fisiopatologia , Técnicas In Vitro , Artéria Mesentérica Superior/citologia , Artéria Mesentérica Superior/metabolismo , Relaxamento Muscular/fisiologia , Músculo Liso Vascular/citologia , Coelhos , Sistemas do Segundo Mensageiro/fisiologia , Resistência Vascular/fisiologiaAssuntos
Proteínas de Transporte/genética , Proteínas de Neoplasias , Receptores de N-Metil-D-Aspartato/genética , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/genética , Humanos , Dados de Sequência MolecularRESUMO
Stretch-inactivated channels (SICs) were identified in single smooth muscle cells freshly dissociated from the stomach of the toad, Bufo marinus. In both cell-attached and excised inside-out patches, negative pressure applied to the extracellular surface of the membrane patch suppressed the activity of SICs. These channels were permeable to cations and were not significantly permeable to Cl-. The current-voltage relationship showed outward rectification in cell-attached patches with high NaCl in the pipette solution (2 mM MgCl2), and the slope conductance at negative potentials was approximately 8 pS under these conditions. When divalent cations were eliminated from the pipette solution, the slope conductance at negative potentials increased to approximately 30 pS. No significant voltage dependence of SIC gating could be observed between -100 mV and 60 mV.
Assuntos
Canais Iônicos/fisiologia , Músculo Liso/fisiologia , Animais , Bufo marinus , Potenciais da Membrana , Músculo Liso/citologia , Estimulação Física , Estômago/citologiaRESUMO
Aluminofluoride (AF) has a variety of biological actions such as activation of GTP binding proteins and inhibition of phosphatases. In the present study, the effects of AF on hyper-polarization- and stretch-activated cationic channels (HA-SACs) were investigated in isolated gastric smooth muscle cells from the toad, Bufo marinus, using the patch-clamp technique. In cell-attached patches extracellular application of AF (20 mM KF plus 20 microM AlCl3) reversibly increased HA-SAC activity without changing its voltage sensitivity. The single channel current amplitude of HA-SACs was not affected during this procedure. The mechanism of AF-induced activation of HA-SACs remains unclear. However, this activation may play a role in contraction of smooth muscle induced by AF.
Assuntos
Compostos de Alumínio , Alumínio/farmacologia , Fluoretos/farmacologia , Canais Iônicos/efeitos dos fármacos , Músculo Liso/fisiologia , Animais , Bufo marinus , Canais Iônicos/fisiologia , Potenciais da Membrana , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Estômago/citologiaRESUMO
The role of the second messenger diacylglycerol (DAG) in mediating muscarinic suppression of M-current, a type of a voltage-gated K+ current that is suppressed by acetylcholine (ACh), was examined in freshly isolated smooth muscle cells from toad stomach. Currents were recorded using a single electrode voltage clamp employing conventional microelectrodes. Extracellular application of 1,2-dioctanoyl-sn-glycerol (DiC8), a synthetic DAG that is a potent activator of protein kinase C (PKC), reversibly suppressed M-current. Current relaxations, representing the voltage-dependent closure of K+ channels underlying M-current, were also decreased by DiC8, although suppression was not always as complete as it was with ACh. In contrast, another DAG analogue, 1,2-dioctanoyl-3-thioglycerol, which has a structure closely related to DiC8 but does not activate PKC, failed to inhibit M-current. Furthermore, M-current induced by the beta-agonist isoproterenol, by a mechanism apparently mediated by adenosine 3',5'-cyclic monophosphate (S. M. Sims, L. H. Clapp, J. V. Walsh, Jr., and J. J. Singer. Pflugers Arch. 417: 291, 1990), was also suppressed by DiC8. Both ACh and DiC8 were found to suppress endogenous and isoproterenol-induced M-current without altering the time course of M-current deactivation, suggesting that these agents act by decreasing the number of channels available to be opened. These results provide evidence that muscarinic regulation of M-current is mediated by DAG.
Assuntos
Acetilcolina/farmacologia , Diglicerídeos/fisiologia , Músculo Liso/efeitos dos fármacos , Estômago/efeitos dos fármacos , Animais , Bufo marinus , Diglicerídeos/farmacologia , Condutividade Elétrica , Isoproterenol/farmacologia , Músculo Liso/citologia , Músculo Liso/fisiologia , Estômago/citologia , Estômago/fisiologiaRESUMO
Calcium entry through voltage-activated Ca2+ channels is important in regulating many cellular functions. Activation of these channels in many cell types results in feedback regulation of channel activity. Mechanisms linking Ca2+ channel activity with its downregulation have been described, but little is known of the events responsible for the enhancement of Ca2+ current that in many cells follows Ca2+ channel activation and an increase in cytoplasmic Ca2+ concentration. Here we investigate how this positive feedback is achieved in single smooth muscle cells. We find that in these cells voltage-activated calcium current is persistently but reversibly enhanced after periods of activation. This persistent enhancement of the Ca2+ current is mediated by activation of calmodulin-dependent protein kinase II because it is blocked when either the rise in cytoplasmic Ca2+ is inhibited or activation of calmodulin-dependent protein kinase II is prevented by specific peptide inhibitors of calcium-calmodulin or calmodulin-dependent protein kinase II itself. This mechanism may be important in different forms of Ca2+ current potentiation, such as those that depend on prior Ca2+ channel activation or are a result of agonist-induced release of Ca2+ from internal stores.
Assuntos
Cálcio/fisiologia , Músculo Liso/fisiologia , Proteínas Quinases/fisiologia , Sequência de Aminoácidos , Animais , Bufo marinus , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Condutividade Elétrica , Técnicas In Vitro , Potenciais da Membrana/fisiologia , Dados de Sequência MolecularRESUMO
Large conductance Ca(2+)-activated K+ channels in rabbit pulmonary artery smooth muscle cells are activated by membrane stretch and by arachidonic acid and other fatty acids. Activation by stretch appears to occur by a direct effect of stretch on the channel itself or a closely associated component. In excised inside-out patches stretch activation was seen under conditions which precluded possible mechanisms involving cytosolic factors, release of Ca2+ from intracellular stores, or stretch induced transmembrane flux of Ca2+ or other ions potentially capable of activating the channel. Fatty acids also directly activate this channel. Like stretch activation, fatty acid activation occurs in excised inside-out patches in the absence of cytosolic constituents. Moreover, the channel is activated by fatty acids which, unlike arachidonic acid, are not substrates for the cyclo-oxygenase or lypoxygenase pathways, indicating that oxygenated metabolites do not mediate the response. Thus, four distinct types of stimuli (cytosolic Ca2+, membrane potential, membrane stretch, and fatty acids) can directly affect the activity of this channel.