RESUMEN
ATP evokes changes in the vascular tone through the activation of P2X and P2Y purinoceptors. To evaluate relative contribution of Ca2+ entry through the L-type voltage-gated calcium channels and Ca2+ -induced Ca2+ -release (CICR) mechanisms in initiation of vascular smooth muscle contraction induced by P2XRs activation, we have applied P2X receptor agonist -meATP (10 microM) and measured changes in phasic isometric tension of endothelium-denuded mesenteric artery rings in the presence of antagonists IP3Rs (60 mcmol/l APB) or RyRs (100 mcmol/l tetracaine) combined with on-off modulation of the L-type calcium channels by nicardipine (5 microM). We found that activation of P2XRs results in Ca2+ release from SR through both IP3Rs and RyRs. In addition, Ca2+ entry via L-type Ca2+ channels also participates in Ca2+ release from SR presumably through CICR mechanism. However, the phasic contractions in the presence of nicardipine were found to be less sensitive to inhibition of IP3Rs than RyRs (47.1 +/- 9.5% and 22.9 +/- 1.4% comparing to 38.3 +/- 9.6% and 518 +/- 7.8% in control solution for IP3R and RyR inhibition, respectively). This finding suggests that Ca2+ entered the cell through L-type Ca2+ channels, has easier access to IP3Rs than to RyRs. This suggestion is further supported by immunostaining IP3Rs and RyRs. Confocal imaging revealed that sub-PM SR elements are enriched with IP3Rs, while RyRs are predominantly located in the central/perinuclear SR elements.
Asunto(s)
Calcio/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Cobayas , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Arterias Mesentéricas/metabolismo , Microscopía Confocal , Músculo Liso Vascular/metabolismo , Nicardipino/farmacología , Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2X , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.
Asunto(s)
Calcio/fisiología , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Contracción Isométrica/fisiología , Miocitos del Músculo Liso/fisiología , Animales , Compuestos de Boro/farmacología , Señalización del Calcio/fisiología , Carbacol/farmacología , Membrana Celular/fisiología , Potenciales Evocados/efectos de los fármacos , Cobayas , Íleon/fisiología , Contracción Isométrica/efectos de los fármacos , Compuestos Macrocíclicos/farmacología , Masculino , Nicardipino/farmacología , Oxazoles/farmacología , Receptores Muscarínicos/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Tetracaína/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: There is little information about the excitatory cholinergic mechanisms of mouse small intestine although this model is important for gene knock-out studies. EXPERIMENTAL APPROACH: Using patch-clamp techniques, voltage-dependent and pharmacological properties of carbachol- or intracellular GTPgammaS-activated cationic channels in mouse ileal myocytes were investigated. KEY RESULTS: Three types of cation channels were identified in outside-out patches (17, 70 and 140 pS). The voltage-dependent behaviour of the 70 pS channel, which was also the most abundantly expressed channel (approximately 0.35 micro(-2)) was most consistent with the properties of the whole-cell muscarinic current (half-maximal activation at -72.3+/-9.3 mV, slope of -9.1+/-7.4 mV and mean open probability of 0.16+/-0.01 at -40 mV; at near maximal activation by 50 microM carbachol). Both channel conductance and open probability depended on the permeant cation in the order: Cs+ (70 pS) >Rb+ (66pS) >Na+ (47 pS) >Li+ (30 pS). External application of divalent cations, quinine, SK&F 96365 or La3+ strongly inhibited the whole-cell current. At the single channel level the nature of the inhibitory effects appeared to be very different. Either reduction of the open probability (quinine and to some extent SK&F 96365 and La3+) or of unitary current amplitude (Ca2+, Mg2+, SK&F 96365, La3+) was observed implying significant differences in the dissociation rates of the blockers. CONCLUSIONS AND IMPLICATIONS: The muscarinic cation current of murine small intestine is very similar to that in guinea-pig myocytes and murine genetic manipulation should yield important information about muscarinic receptor transduction mechanisms.
Asunto(s)
Íleon/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores Muscarínicos/fisiología , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/fisiología , Íleon/citología , Masculino , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Distribution and enzymatic activity of haem oxygenase (HO) was investigated in the stomach of healthy guinea pigs or animals subjected to in vivo cobalt-induced oxidative stress. The physiological role of HO-1 and HO-2 isoenzymes in fundic and antral area of the stomach was assessed by studying the action of HO substrate--hemin--on ionic currents of single smooth muscle cells. The data obtained suggest that HO-1 induction might serve in the guinea-pig stomach as genetically determined defense mechanism aimed to combat toxic stress-related pathology in order to preserve the functional performance of the organ.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hemo-Oxigenasa 1/genética , Músculo Liso/fisiología , Estrés Oxidativo/fisiología , Estómago/enzimología , Animales , Cobalto/farmacología , Electrofisiología , Cobayas , Hemo Oxigenasa (Desciclizante)/fisiología , Hemo-Oxigenasa 1/fisiología , Hemina/farmacología , Masculino , Músculo Liso/citología , Potasio/metabolismoRESUMEN
Interstitial cells of Cajal are believed to play an important role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recently cells with similar morphological characteristics have been found in the wall of blood vessels such as rabbit portal vein and guinea pig mesenteric artery. These non-contractile cells are characterised by the presence of numerous processes and were easily detected in the wall of the rabbit portal vein by staining with methylene blue or by antibodies to the marker of Interstitial Cells of Cajal c-kit. These vascular cells have been termed "interstitial cells" by analogy with interstitial cells found in the gastrointestinal tract. Freshly dispersed interstitial cells from rabbit portal vein and guinea pig mesenteric artery displayed various Ca2+-release events from endo/sarcoplasmic reticulum including fast localised Ca2+ transients (Ca2+ sparks) and longer and slower Ca2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca2+ oscillations associated with membrane depolarisations, which suggests that in this vessel interstitial cells may act as pacemakers for smooth muscle cells. The function of interstitial cells from the mesenteric arteries is yet unknown. This article reviews some of the recent findings regarding interstitial cells from blood vessels obtained by our laboratory using electron microscopy, immunohistochemistry, tight-seal patch-clamp recording, and fluorescence confocal imaging techniques.
Asunto(s)
Vasos Sanguíneos/citología , Células del Tejido Conectivo/citología , Animales , Relojes Biológicos/fisiología , Señalización del Calcio/fisiología , Comunicación Celular/fisiología , Células del Tejido Conectivo/fisiología , Células del Tejido Conectivo/ultraestructura , Cobayas , Humanos , Arterias Mesentéricas/citología , Arterias Mesentéricas/ultraestructura , Ratones , Microscopía Electrónica , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Vena Porta/citología , Vena Porta/ultraestructura , ConejosRESUMEN
A rise in intracellular ionised calcium concentration ([Ca(2+)](i)) at sites adjacent to the contractile proteins is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques with special accent on the fluorescence confocal microscopy and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for study of the relationship between the structural organisation of the living smooth muscle myocyte and the features of calcium signalling at subcellular level. Applying fluorescent confocal microscopy and tight-seal recording of transmembrane ion currents to freshly isolated vascular myocytes we have demonstrated that: (1) Ca(2+) sparks originate from clustered opening of ryanodine receptors (RyRs) and build up a cell-wide increase in [Ca(2+)](i) upon myocyte excitation; (2) spontaneous Ca(2+) sparks occurred at the highest rate at certain preferred locations, frequent discharge sites (FDS), which are associated with a prominent portion of the sarcoplasmic reticulum (SR) located close to the cell membrane; (3) Ca(2+)-dependent K(+) and Cl(-) channels sense the local changes in [Ca(2+)](i) during a calcium spark and thereby couple changes in [Ca(2+)](i) within a microdomain to changes in the membrane potential, thus affecting excitability of the cell; (4) an intercommunication between RyRs and inositol trisphosphate receptors (IP(3)Rs) is one of the important determinants of intracellular calcium dynamics that, in turn, can modulate the cell membrane potential through differential targeting of calcium dependent membrane ion channels. Furthermore, using immunohystochemical approaches in combination with confocal imaging we identified non-contractile cells closely resembling interstitial cells (ICs) of Cajal (which are considered to be pacemaker cells in the gut) in the wall of portal vein and mesenteric artery. Using electron microscopy, tight-seal recording and fluorescence confocal imaging we obtained information on the morphology of ICs and their possible coupling to smooth muscle cells (SMCs), calcium signalling in ICs and their electrophysiological properties. The functions of these cells are not yet fully understood; in portal vein they may act as pacemakers driving the spontaneous activity of the muscle; in artery they may have other a yet unsuspected functions.
Asunto(s)
Vasos Sanguíneos/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Animales , Vasos Sanguíneos/metabolismo , Calcio/metabolismo , Arterias Mesentéricas/citología , Arterias Mesentéricas/metabolismo , Vena Porta/citología , Vena Porta/metabolismoRESUMEN
After enzymatic dispersion of the muscle of the guinea-pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non-propagating) intracellular calcium ([Ca(2+)](i)) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio-temporal characteristics (full width and full duration at half-maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell-wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium-free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2-APB (100 microm), ryanodine (10 or 50 microm) or U-73122 (1 microm). 2-APB (100 microm), xestospongin C (XeC, 10 microm) or U-73122 (1 microm) blocked both spontaneous localized calcium release and localized release stimulated by 10 microm carbachol. Ryanodine (50 microm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.
Asunto(s)
Calcio/metabolismo , Fundus Gástrico/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Compuestos de Boro/farmacología , Calcio/administración & dosificación , Canales de Calcio , Carbacol/farmacología , Relación Dosis-Respuesta a Droga , Estrenos/farmacología , Fundus Gástrico/citología , Fundus Gástrico/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Cobayas , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato , Compuestos Macrocíclicos , Microscopía Confocal , Miocitos del Músculo Liso/efectos de los fármacos , Neomicina/farmacología , Oxazoles/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Pirrolidinonas/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Rianodina/farmacología , Programas Informáticos , Distribución Tisular , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
(1) The effects on the whole-cell carbachol-induced muscarinic cationic current (mIcat) of antibodies against the alpha-subunits of various G proteins, as well as the effect of a Gbetagamma subunit, were studied in single guinea-pig ileal smooth muscle cells voltage-clamped at -50 mV. Ionized intracellular calcium concentration, [Ca(2+)](i), was clamped at 100 nM using a 1,2-bis(2-aminophenoxyl-ethane-N,N,N',N'-tetraacetic acid)/Ca(2+) mixture. (2) Application of ascending concentrations of carbachol (1-300 micro M) activated mIcat (mean amplitude 0.83 nA at 300 micro M carbachol; EC(50) 8 micro M; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of G(i3)/G(o) or G(o) antibodies resulted in about a 70% depression of the maximum response without change in the EC(50) value. In contrast, antibodies against alpha-subunits of G(i1), G(i1)/G(i2), G(i3), G(q)/G(11) or G(s) protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gbeta or infusion of the Gbetagamma subunit itself had no effect on mIcat. (3) If cells were exposed briefly to carbachol (50 or 100 micro M) at early times (<3 min) after infusion of antibodies to Galpha(i3)/Galpha(o) or to Galpha(o) had begun, carbachol responses remained unchanged even after 20-60 min; that is, the depression of mIcat by these antibodies was prevented. (4) These data show that Galpha(o) protein couples the muscarinic receptor to the cationic channel in guinea-pig ileal longitudinal smooth muscle and that Gbetagamma is not involved. They also show that prior activation of the muscarinic receptor presumably causes a long-lasting postactivation change of the G protein, which is not reflected in mIcat, but acts to hinder antibody binding.
Asunto(s)
Anticuerpos/farmacología , Especificidad de Anticuerpos , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Subunidades beta de la Proteína de Unión al GTP/fisiología , Subunidades gamma de la Proteína de Unión al GTP/fisiología , Canales Iónicos/metabolismo , Músculo Liso/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Carbacol/farmacología , Cationes , Relación Dosis-Respuesta a Droga , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas de Unión al GTP/metabolismo , Cobayas , Íleon/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso/efectos de los fármacosRESUMEN
Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40-70 microm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 microm, and spindle-shaped cells from 40 to 300 microm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90-150 microm). The cell membrane capacitance was 46.5+/-2.2 pF in SMCs, 39.7+/-2.4 pF in spindle-shaped ICs and 27.8+/-0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca(2+) sparks and Ca(2+) waves. Ca(2+) waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca(2+)](i) transients (lasting about 2s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1-10 microM), caffeine (1-10 mM) or high-K(+) solution (60mM) led to a rise of [Ca(2+)](i) in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current-clamp conditions was -46.5+/-2.0 mV in spindle-shaped ICs and -45.6+/-2.7 mV in SMCs. Under voltage-clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca(2+) current, a set of K(+) currents, including spontaneous transient outward currents (STOCs) but no Na(+) current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Vena Porta/citología , Vena Porta/metabolismo , Compuestos de Anilina , Animales , Cafeína/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Membrana Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Capacidad Eléctrica , Técnica del Anticuerpo Fluorescente , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Azul de Metileno , Músculo Liso Vascular/efectos de los fármacos , Norepinefrina/farmacología , Vena Porta/efectos de los fármacos , Potasio/metabolismo , Potasio/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Conejos , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , XantenosRESUMEN
Although smooth muscle cells are not organized in sarcomeres, as are striated muscles, nevertheless Ca2+ for contraction is released from the sarcoplasmic reticulum (SR) at certain preferred sites. These sites commonly discharge packets of Ca2+ spontaneously and have been called frequent discharge sites (FDSs). Each spontaneous release of a Ca2+ packet usually leads to a burst of openings of Ca2+-activated K+ channels in the cell membrane which produces a spontaneous transient outward current (STOC) in smooth muscle cells under voltage clamp. When fluorescent Ca2+ indicators such as Fluo-3 became available, the spontaneous transient increases in [Ca2+]i produced by Ca2+ packets released from the SR were also detected in cardiac muscle as flashes of fluorescence or 'sparks'. Sparks in smooth muscle consist of smaller Ca2+ packets that can give rise to 'microsparks'. In some smooth muscles which have Ca2+-activated Cl- channels, STICs (spontaneous transient inward currents) are also found to be associated with sparks. FDSs have been found to be important initiating sites for a Ca2+ wave in response to an action potential or in response to receptor activation and possibly other stimuli, such as stretch. In both cases Ca2+-induced Ca2+ release seems to be crucially involved.
Asunto(s)
Calcio/fisiología , Contracción Muscular/fisiología , Músculo Liso/fisiología , Animales , Íleon/fisiología , Mamíferos , Potenciales de la Membrana/fisiología , Relajación Muscular/fisiología , Músculo Liso Vascular/fisiología , Sarcómeros/fisiologíaRESUMEN
In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca(2+)-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca(2+) indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca(2+)-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca(2+) sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F(0)) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 microm) for the 230 localised Ca(2+)-release events analysed. The existence of two populations of Ca(2+)-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca(2+)-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3-5 min exposure to 1-5 microM nicardipine, but were abolished after 10-15 min exposure to 50-100 microM ryanodine, 0.1 microM thapsigargin or 10 microM cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, 10 microM xestospongin C or 30 microM 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 microM U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca(2+)-release domains giving rise to spontaneous [Ca(2+)](i) waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP(3) receptors to Ca(2+), which can give rise to 'loose coupling' between release sites. These results suggest that activation of IP(3) receptors (due to the tonic activity of PLC and ongoing production of IP(3)) recruits neighbouring domains of ryanodine receptors, leading to larger Ca(2+) releases and saltatory propagation of [Ca(2+)](i) waves in portal vein myocytes.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Células Musculares/metabolismo , Vena Porta/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Compuestos de Boro/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Estrenos/farmacología , Receptores de Inositol 1,4,5-Trifosfato , Membranas Intracelulares/metabolismo , Compuestos Macrocíclicos , Masculino , Oxazoles/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Vena Porta/citología , Pirrolidinonas/farmacología , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The role of heme oxygenase reaction products in modulation of stomach fundus excitability was studied. The presence of constitutive heme oxygenase 2 was verified in myenteric ganglia by immunohistochemistry. The role of inducible heme oxygenase isoenzyme was investigated after invivo treatment of animals with CoCl2 (80 mg kg-1 b.w) injected subcutaneously 24 h before they were killed. This treatment resulted in increased production of bilirubin and positive staining for the inducible isoform in stomach smooth muscle and vast induction in the liver. In both control and treated animals haemin, applied to the bath as a substrate of heme oxygenase caused significant decrease of prostaglandin F2alpha-induced tone, and ameliorated the relaxatory response of the fundic strips to electrical field stimulation. Both effects were antagonized by Sn-protoporphyrin IX, competitive heme oxygenase inhibitor, and were found to be neuronally dependent. In single freshly isolated smooth muscle cells from control animals haemin caused a concentration-dependent increase of the whole cell K+ currents, which was not affected by Sn-protoporphyrin IX, cyclic guanosine monophosphate (cGMP)-dependent protein kinase or guanylyl cyclase antagonists, but was reversed by various antioxidants and abolished by an NO scavenger. In cells from treated animals the K+ current increasing effect of haemin did not depend on the presence of antioxidants, but was abolished by protein kinase G and guanylyl cyclase inhibitors, depletors of intracellular Ca2+ pools or Sn-protoporphyrin IX. Biliverdin did not affect contraction or ionic currents. Thus, this is the first study demonstrating that heme oxygenase is an inducible enzyme in guinea-pigs, which exerts a modulatory role on gastric smooth muscle excitability via carbon monoxide production.
Asunto(s)
Fundus Gástrico/enzimología , Hemo Oxigenasa (Desciclizante)/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/enzimología , Animales , Antioxidantes/farmacología , Calcio/metabolismo , Cloruro de Calcio/farmacología , Células Cultivadas , Dinoprost/biosíntesis , Inhibidores Enzimáticos/farmacología , Fundus Gástrico/efectos de los fármacos , Cobayas , Hemo-Oxigenasa 1 , Hemina/farmacología , Isoenzimas/fisiología , Masculino , Metaloporfirinas/farmacología , Músculo Liso/efectos de los fármacos , Potasio/metabolismo , Protoporfirinas/farmacologíaRESUMEN
1. The abilities of muscarinic agonists (arecoline, bethanechol, carbachol, McN-A343, methacholine, pilocarpine) to inhibit isoprenaline-induced cyclic AMP production in chopped fragments (via M(2) receptors), and to evoke cationic current (I(cat)) (via M(2) receptors) or calcium store release (via M3 receptors) in enzyme-dispersed, single voltage-clamped cells from longitudinal smooth muscle of the guinea-pig small intestine were examined. 2. All muscarinic agonists (1 - 300 microM) examined inhibited isoprenaline (1 microM)-induced accumulation of cyclic AMP, the IC(50) varying from 52 to 248 microM. However, their relative potencies to evoke this M(2) effect were not significantly correlated with their ability to evoke I(cat), also a M(2) effect, whether or not calcium stores were depleted; pilocarpine and McN-A343 inhibited the I(cat) response to carbachol. 3. Muscarinic agonists (concentration 300 or 1000 microM), except pilocarpine and McN-A343 which were ineffective, evoked Ca(2+)-activated K(+) current (I(K-Ca)) resulting from Ca(2+) store release (M(3) effect). Their effectiveness was tested by estimating residual stored calcium by subsequent application of caffeine (10 mM). The relative potencies to evoke Ca(2+) store release (M(3)) and for I(cat) activation (M(2)) were closely correlated (P<0.001). 4. These data might be explained if M(2)-mediated adenylyl cyclase inhibition and I(cat) activation involve different G proteins, or involve different populations of M(2) receptors. The observed correlation of agonist potency between I(cat) activation and Ca(2+) store release supports the proposal (Zholos & Bolton, 1997) that M(3) activation can potentiate M(2)-cationic channel coupling through Ca(2+)-independent mechanisms.
Asunto(s)
Intestino Delgado/citología , Agonistas Muscarínicos/farmacología , Músculo Liso/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Electrofisiología , Cobayas , Intestino Delgado/fisiología , Masculino , Músculo Liso/metabolismo , Músculo Liso/fisiología , Canales de Potasio Calcio-Activados/fisiología , Receptor Muscarínico M2 , Receptor Muscarínico M3RESUMEN
Localized Ca(2+)-release events, Ca(2+)sparks, have been suggested to be the 'elementary building blocks' of the calcium signalling system in all types of muscles. In striated muscles these occur at regular intervals along the fibre corresponding to the sarcomeric structures which do not exist in smooth muscle. We showed previously that in visceral and vascular myocytes Ca(2+)sparks occurred much more frequently at certain sites (frequent discharge sites [FDSs]). In this paper, we have related the position of FDSs to the distribution of the sarcoplasmic reticulum in the same living myocyte. The three-dimensional distribution of the SR in freshly isolated rabbit portal vein myocytes was visualized by means of high-resolution confocal imaging after staining with DiOC(6)and/or BODIPY TR-X ryanodine. Both fluorochromes revealed a similar staining pattern indicating a helical arrangement of well-developed superficial SR which occupied about 6% of the cell volume. Computing the frequency of spontaneous Ca(2+)sparks detected by means of fluo-4 fluorescence revealed that in about 70% of myocytes there was only one major FDS located on a prominent portion of superficial SR network usually within 1-2 microm of the nuclear envelope, although a few sparks occurred at other sites scattered generally in superficial locations throughout the cell. Polarized mitochondria were readily identified by accumulation of tetramethylrhodamine ethyl ester (TMRE). These were closely associated with the SR network in extra-nuclear regions. TMRE staining, however, failed to reveal any mitochondria near the FDS-related SR element. When observed, propagating [Ca(2+)](i)waves and associated myocyte contractions were initiated at FDSs. This study provide first insight into the three-dimensional arrangement of the SR in living smooth muscle cells and relates the peculiarity of the structural organization of the myocyte to the features of Ca(2+)signalling at subcellular level.
Asunto(s)
Calcio/metabolismo , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Músculo Liso Vascular/metabolismo , Retículo Sarcoplasmático/metabolismo , Compuestos de Anilina , Animales , Compuestos de Boro , Carbocianinas , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador/métodos , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Liso Vascular/citología , Compuestos Organometálicos , Vena Porta/citología , Conejos , Rianodina , XantenosRESUMEN
Using whole-cell patch-clamp recording techniques, we have examined voltage-gated ion currents in a cultured human intestinal smooth muscle cell line (HISM). Experiments were performed at room temperature on cells after passages 16 and 17. Two major components of the whole-cell current were a tetraethylammonium-sensitive (IC50 = 9 mM), iberiotoxin-resistant, delayed rectifier K+ current and a Na+ current inhibited by tetrodotoxin (IC50 A 100 nM). No measurable inward current via voltage-gated Ca2+ channels could be detected in these cells even with 10 mM Ca2+ or Ba2+ in the external solution. No current attributable to calcium-activated K+ channels was found and no cationic current in response to muscarinic receptor activation was present. In divalent cation-free external solution two additional currents were activated: an inwardly rectifying hyperpolarization-activated current, I(HA), and a depolarization-activated current, I(DA) x I(HA) and I(DA) could be carried by several monovalent cations; the sizes of currents in descending order were: K+ > Cs+ > Na+ for I(HA) and Na+ > K+ >> Cs+ for I(DA). I(HA) was activated and deactivated instantaneously and showed no inactivation whereas I(DA) was activated, inactivated and deactivated within tens of milliseconds. These currents were inhibited by external calcium with an IC50 of 0.3 microM for I(DA) and an IC50 of 20 microM for I(HA). Cyclopiazonic acid (CPA) induced an outward, but not an inward current. SK&F 96365, a blocker of store-operated Ca2+ channels, suppressed I(DA) with a half-maximal inhibitory concentration of 9 microM but was ineffective in inhibiting I(HA) at concentrations up to 100 microM. Gd3+ and La3+ strongly suppressed I(DA) at 1 and 10 microM, respectively and were less effective in blocking I(HA) (complete inhibition required a concentration of 100 microM for both). Carbachol at 10-100 microM evoked about a 3-fold increase in I(HA) amplitude and completely abolished I(DA). We conclude that I(HA) and I(DA) are Ca2+-blockable cationic currents with different ion selectivity profiles that are carried by different channels. I(DA) shows novel voltage-dependent properties for a cationic current.
Asunto(s)
Mucosa Intestinal/metabolismo , Canales Iónicos/fisiología , Músculo Liso/metabolismo , Cationes/metabolismo , Línea Celular , Conductividad Eléctrica , Humanos , Intestinos/citología , Músculo Liso/citología , Técnicas de Placa-Clamp , Canales de Sodio/efectos de los fármacos , Canales de Sodio/fisiología , Tetrodotoxina/farmacologíaRESUMEN
The effects of SK&F 96365 on cationic current evoked either by activating muscarinic receptors with carbachol or by intracellularly applied GTPgammaS (in the absence of carbachol) were studied using patch-clamp recording techniques in single guinea-pig ileal smooth muscle cells. SK&F 96365 reversibly inhibited the muscarinic receptor cationic current in a concentration-, time- and voltage-dependent manner producing concomitant alteration of the steady-state I-V relationship shape which could be explained by assuming that increasing membrane positivity increased the affinity of the blocker. The inhibition was similar for both carbachol- and GTPgammaS-evoked currents suggesting that the cationic channel rather than the muscarinic receptor was the primary site of the SK&F 96365 action. Increased membrane positivity induced additional rapid inhibition of the cationic current by SK&F 96365 which was more slowly relieved during membrane repolarization. Both the inhibition and disinhibition time course could be well fitted by a single exponential function with the time constants decreasing with increasing positivity for the inhibition (e-fold per about 12 mV) and approximately linearly decreasing with increasing negativity for the disinhibition. At a constant SK&F 96365 concentration, the degree of cationic current inhibition was a sigmoidal function of the membrane potential with a potential of half-maximal increase positive to about +30 mV and a slope factor of about -13 mV. Increasing the duration of voltage steps at -80 or at 80 mV, increased the percentage inhibition; the degree of inhibition was almost identical at both potentials providing evidence that the same cationic channel was responsible for the cationic current both at negative and at positive potentials. It is concluded that the distinctive and unique mode of SK&F 96365 action on the muscarinic receptor cationic channel is a valuable tool in future molecular biology studies of this channel.
Asunto(s)
Imidazoles/farmacología , Canales Iónicos/antagonistas & inhibidores , Antagonistas Muscarínicos/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Receptores Muscarínicos/fisiología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Carbacol/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Cobayas , Íleon/citología , Íleon/efectos de los fármacos , Íleon/fisiología , Canales Iónicos/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/citología , Receptores Muscarínicos/metabolismoRESUMEN
Ionized calcium plays a central role as a second messenger in a number of physiologically important processes determining smooth muscle function. To regulate a wide range of cellular activities the mechanisms of subcellular calcium signalling should be very diverse. Recent progress in development of visible light-excitable fluorescent dyes with high affinity for Ca2+ (such as oregon green 488 BAPTA indicators, fluo-3 and fura red) and confocal laser scanning microscopy provides an opportunity for direct visualization of subcellular Ca2+ signalling and reveals that many cell function are regulated by the microenvironment within small regions of the cytoplasm ('local control' concept). Here confocal imaging is used to measure and locate changes in [Ca2+]i on a subcellular level in response to receptor stimulation in visceral myocytes. We show that stimulation of muscarinic receptors in ileal myocytes with carbachol leading to activation of inositol 1,4,5-trisphosphate receptors (IP3Rs) accelerates the frequency of spontaneous calcium sparks (discharged via ryanodine receptors, RyRs) and gives rise to periodic propagating Ca2+ waves oscillating with a frequency similar to that of carbachol-activated cationic current oscillations. Furthermore, by combining the whole-cell patch clamp technique with simultaneous confocal imaging of [Ca2+]i in voltage-clamped vascular myocytes we demonstrate that calcium sparks may lead to the opening of either Ca2+-activated Cl- channels or Ca2+-activated K+ channels, and the discharge of a spontaneous transient inward current (STIC) or a spontaneous transient outward current (STOC), respectively.
Asunto(s)
Señalización del Calcio , Animales , Carbacol/farmacología , Canales de Cloruro/fisiología , Colorantes Fluorescentes , Cobayas , Íleon/citología , Masculino , Potenciales de la Membrana/fisiología , Arteria Mesentérica Superior/citología , Microscopía Confocal , Microscopía Fluorescente , Contracción Muscular/fisiología , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio/fisiologíaRESUMEN
The main contributors to increases in [Ca2+]i and tension are the entry of Ca2+ through voltage-dependent channels opened by depolarization or during action potential (AP) or slow-wave discharge, and Ca2+ release from store sites in the cell by the action of IP3 or by Ca(2+)-induced Ca(2+)-release (CICR). The entry of Ca2+ during an AP triggers CICR from up to 20 or more subplasmalemmal store sites (seen as hot spots, using fluorescent indicators); Ca2+ waves then spread from these hot spots, which results in a rise in [Ca2+]i throughout the cell. Spontaneous transient releases of store Ca2+, previously detected as spontaneous transient outward currents (STOCs), are seen as sparks when fluorescent indicators are used. Sparks occur at certain preferred locations--frequent discharge sites (FDSs)--and these and hot spots may represent aggregations of sarcoplasmic reticulum scattered throughout the cytoplasm. Activation of receptors for excitatory signal molecules generally depolarizes the cell while it increases the production of IP3 (causing calcium store release) and diacylglycerols (which activate protein kinases). Activation of receptors for inhibitory signal molecules increases the activity of protein kinases through increases in cAMP or cGMP and often hyperpolarizes the cell. Other receptors link to tyrosine kinases, which trigger signal cascades interacting with trimeric G-protein systems.
Asunto(s)
Fenómenos Fisiológicos del Sistema Digestivo , Motilidad Gastrointestinal/fisiología , Músculo Liso/fisiología , Animales , Calcio/metabolismo , Sistema Digestivo/citología , Cobayas , Humanos , Músculo Liso/citología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/metabolismo , Transducción de Señal/fisiologíaRESUMEN
1. Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo-3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea-pig vas deferens or urinary bladder. 2. Images obtained every 8 ms, during stepping from -60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 micrometer diameter) 'hot spots', over 95 % of which were located within 1.5 micrometer of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places. 3. Cd2+ or verapamil abolished both hot spots and Ca2+-activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+-induced Ca2+ release (CICR). 4. Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to -20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up-stroke of an evoked action potential under current clamp. 5. It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+-dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.
Asunto(s)
Calcio/metabolismo , Músculo Liso/metabolismo , Canales de Potasio/metabolismo , Vejiga Urinaria/metabolismo , Conducto Deferente/metabolismo , Potenciales de Acción/efectos de los fármacos , Compuestos de Anilina , Animales , Bloqueadores de los Canales de Calcio/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Estimulación Eléctrica , Electrofisiología , Colorantes Fluorescentes , Cobayas , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Canales de Potasio/agonistas , Vejiga Urinaria/citología , Vejiga Urinaria/efectos de los fármacos , Conducto Deferente/citología , Conducto Deferente/efectos de los fármacos , XantenosRESUMEN
The effect of the lipophilic quaternary ion, tetraphenylphosphonium, on membrane potential of segments of rat small mesenteric artery and on the current in single voltage-clamped smooth muscle cells from rabbit portal vein was studied. In rat small mesenteric artery, tetraphenylphosphonium (1-30 microM) caused membrane depolarization of approximately 23 mV and decreased or abolished the hyperpolarization induced by the KATP channel opener, levcromakalim (0.1-3 microM). In rabbit portal vein K+ currents induced by levcromakalim (10 microM) or pinacidil (10 microM) were completely inhibited by tetraphenylphosphonium (IC50 0.5 microM). The results show that tetraphenylphosphonium antagonizes the KATP current induced by K+ channel openers in vascular smooth muscle possibly by acting on the KATP channel itself.