RESUMEN
The alveolar-capillary barrier is composed of epithelial and endothelial cells interacting across a fibrous extracelluar matrix (ECM). Although remodeling of the ECM occurs during several lung disorders, it is not known how fiber structure and mechanics influences cell injury during cyclic airway reopening as occurs during mechanical ventilation (atelectrauma). We have developed a novel in vitro platform that mimics the micro/nano-scale architecture of the alveolar microenvironment and have used this system to investigate how ECM microstructural properties influence epithelial cell injury during airway reopening. In addition to epithelial-endothelial interactions, our platform accounts for the fibrous topography of the basal membrane and allows for easy modulation of fiber size/diameter, density and stiffness. Results indicate that fiber stiffness and topography significantly influence epithelial/endothelial barrier function where increased fiber stiffness/density resulted in altered cytoskeletal structure, increased tight junction (TJ) formation and reduced barrier permeability. However, cells on rigid/dense fibers were also more susceptible to injury during airway reopening. These results indicate that changes in the mechanics and architecture of the lung microenvironment can significantly alter cell function and injury and demonstrate the importance of implementing in vitro models that more closely resemble the natural conditions of the lung microenvironment.
Asunto(s)
Barrera Alveolocapilar/fisiología , Matriz Extracelular/metabolismo , Técnicas In Vitro , Microtecnología , Atelectasia Pulmonar/etiología , Atelectasia Pulmonar/metabolismo , Actinas/metabolismo , Células Epiteliales Alveolares/metabolismo , Materiales Biomiméticos , Células Cultivadas , Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Humanos , Microtecnología/instrumentación , Microtecnología/métodos , Atelectasia Pulmonar/patología , Uniones EstrechasRESUMEN
The personality dimension of schizotypy is well established, and schizotypal traits can be taken to represent a proneness toward developing psychosis. Yet, there are competing theories about the latent structure of schizotypy. More specifically, there is controversy over the extent to which this propensity toward psychosis is present only in a small proportion of the population, or whether it is spread dimensionally throughout the general community. On the basis of accumulating research findings the present article argues for a fully dimensional model of schizotypy. It describes recent neurobiological, neuropsychological, social and environmental evidence supporting the idea that schizotypy in healthy populations, and disorders on the schizophrenia spectrum are fundamentally linked. Directions for further research are also considered.
Asunto(s)
Trastornos Psicóticos/fisiopatología , Esquizofrenia/fisiopatología , Trastorno de la Personalidad Esquizotípica/fisiopatología , Animales , Humanos , Inventario de Personalidad , Factores de Riesgo , Encuestas y CuestionariosRESUMEN
Personalized cancer therapies drive the need for devices that rapidly and accurately segregate cancer cells from solid tumors. One potential sorting strategy is to segregate populations of cells based on their relative strength of adhesion. To investigate the effect of surface hydrophilicity and cell phenotype on adhesion, primary human breast skin fibroblasts and keratinocytes and MCF-7 breast cancer cells were seeded onto air and CF(4) plasma-treated nanofibers followed by exposure to three shear stresses (200, 275 and 350 dynes per cm(2)) 1 hour after inoculation. No difference in strength of adhesion was measured in either fibroblasts or keratinocytes on either plasma treated-surface: all exhibited >60% of the initial cell count after a 5 minute exposure to 350 dynes per cm(2) of shear stress. In contrast, a significant difference between relative strength of adhesion on air versus CF(4) plasma-treated surfaces was observed for MCF-7 cells: 26% and 6.6% of cells remained on the air and CF(4) plasma-treated surfaces, respectively. The ability to sort this cancer cell line from two non-cancerous primary human cells was evaluated by inoculating a mixture of all three cell types simultaneously onto CF(4) treated nanofibers followed by 1 hour of culture and exposure to 350 dynes per cm(2) shear stress. The majority of MCF-7 cells were removed (0.7% remained) while a majority of fibroblasts and keratinocytes remained adhered (74 and 57%). Post-sorted MCF-7 viability and morphology remained unchanged, preserving the possibility of post-separation and analysis. These data suggest that the plasma treatment of electrospun scaffolds provides a tool useful in sorting cancer cells from a mixed cell population based on adhesion strength.
Asunto(s)
Neoplasias de la Mama/patología , Adhesión Celular/fisiología , Fluorocarburos/química , Nanofibras/química , Neoplasias de la Mama/ultraestructura , Supervivencia Celular/fisiología , Femenino , Humanos , Células MCF-7 , Microfluídica , Microscopía Electrónica de Rastreo , Microscopía de Contraste de Fase , Propiedades de SuperficieRESUMEN
UNLABELLED: BACKGROUND; Decreased gallbladder smooth muscle (GBSM) contractility is a hallmark of cholesterol gallstone disease, but the interrelationship between lithogenicity, biliary stasis, and inflammation are poorly understood. We studied a mouse model of gallstone disease to evaluate the development of GBSM dysfunction relative to changes in bile composition and the onset of sterile cholecystitis. METHODS: BALB/cJ mice were fed a lithogenic diet for up to 8 weeks, and tension generated by gallbladder muscle strips was measured. Smooth muscle Ca(2+) transients were imaged in intact gallbladder. KEY RESULTS: Lipid composition of bile was altered lithogenically as early as 1 week, with increased hydrophobicity and cholesterol saturation indexes; however, inflammation was not detectable until the fourth week. Agonist-induced contractility was reduced from weeks 2 through 8. GBSM normally exhibits rhythmic synchronized Ca(2+) flashes, and their frequency is increased by carbachol (3 µm). After 1 week, lithogenic diet-fed mice exhibited disrupted Ca(2+) flash activity, manifesting as clustered flashes, asynchronous flashes, or prolonged quiescent periods. These changes could lead to a depletion of intracellular Ca(2+) stores, which are required for agonist-induced contraction, and diminished basal tone of the organ. Responsiveness of Ca(2+) transients to carbachol was reduced in mice on the lithogenic diet, particularly after 4-8 weeks, concomitant with appearance of mucosal inflammatory changes. CONCLUSIONS & INFERENCES: These observations demonstrate that GBSM dysfunction is an early event in the progression of cholesterol gallstone disease and that it precedes mucosal inflammation.
Asunto(s)
Bilis/química , Colelitiasis/fisiopatología , Colesterol/efectos adversos , Cálculos Biliares/fisiopatología , Músculo Liso/fisiopatología , Animales , Colecistitis/etiología , Colecistitis/patología , Colecistitis/fisiopatología , Colelitiasis/etiología , Colelitiasis/patología , Colesterol en la Dieta/efectos adversos , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Vesícula Biliar/patología , Vesícula Biliar/fisiopatología , Cálculos Biliares/complicaciones , Cálculos Biliares/patología , Inmunohistoquímica , Lípidos , Masculino , Ratones , Ratones Endogámicos BALB C , Contracción Muscular/fisiología , Músculo Liso/patologíaRESUMEN
BACKGROUND AND PURPOSE: Controlling vascular tone involves K(+) efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K(Ca)2.3 and K(Ca)3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling. EXPERIMENTAL APPROACH: Transgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K(Ca)2.3 and K(Ca)3.1 expression by immunohistochemistry and RT-PCR. K(Ca) currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K(Ca)3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry. KEY RESULTS: K(Ca)3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K(Ca)2.3 and K(Ca)3.1 mRNA expression. With 200 nM [Ca(2+)](i) , the K(Ca)2.1-2.3/K(Ca)3.1 opener NS309 increased whole-cell currents. CyPPA, a K(Ca)2.2/K(Ca)2.3 opener, was without effect. With 1 µM [Ca(2+)](i) , the K(Ca)3.1 inhibitor TRAM-34 reduced currents whereas apamin (K(Ca)2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K(Ca)3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K(Ca)3.1 and the large-conductance K(Ca) channel, K(Ca)1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%. CONCLUSION AND IMPLICATIONS: Astrocytes express functional K(Ca)3.1 channels, and these contribute to neurovascular coupling.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Animales , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Calcio/metabolismo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Inmunohistoquímica/métodos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
The nuclear factor of activated T-cells (NFAT) is a Ca(2+)-dependent transcription factor that has been reported to regulate the expression of smooth muscle contractile proteins and ion channels. Here we report that large conductance Ca(2+)-sensitive potassium (BK) channels and voltage-gated K(+) (K(V)) channels may be regulatory targets of NFATc3 in urinary bladder smooth muscle (UBSM). UBSM myocytes from NFATc3-null mice displayed a reduction in iberiotoxin (IBTX)-sensitive BK currents, a decrease in mRNA for the pore-forming alpha-subunit of the BK channel, and a reduction in BK channel density compared with myocytes from wild-type mice. Tetraethylammonium chloride-sensitive K(V) currents were elevated in UBSM myocytes from NFATc3-null mice, as was mRNA for the Shab family member K(V)2.1. Despite K(V) current upregulation, bladder strips from NFATc3-null mice displayed an elevated contractile response to electrical field stimulation relative to strips from wild-type mice, but this difference was abrogated in the presence of the BK channel blocker IBTX. These results support a role for the transcription factor NFATc3 in regulating UBSM contractility, primarily through an NFATc3-dependent increase in BK channel activity.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Factores de Transcripción NFATC/metabolismo , Vejiga Urinaria/metabolismo , Animales , Estimulación Eléctrica , Técnicas In Vitro , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Potenciales de la Membrana , Ratones , Ratones Noqueados , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Factores de Transcripción NFATC/deficiencia , Factores de Transcripción NFATC/genética , Péptidos/farmacología , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , ARN Mensajero/metabolismo , Canales de Potasio Shab/metabolismo , Tetraetilamonio/farmacología , Vejiga Urinaria/citología , Vejiga Urinaria/efectos de los fármacosRESUMEN
Small-conductance Ca(2+)-activated K(+) (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing beta-galactosidase (beta-gal) under the direction of the SK2 promoter (SK2 beta-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2(-/-) mice) to assess SK2 function. In SK2 beta-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2(+/+)) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC(50) = 0.16 nM), whereas phasic contractions of SK2(-/-) strips were unaffected. Nerve-mediated contractions of SK2(+/+) UBSM strips were also increased by apamin, an effect absent in SK2(-/-) strips. Apamin increased the sensitivity of SK2(+/+) UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 +/- 4 Hz, n = 6; apamin, 12 +/- 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2(-/-) UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.
Asunto(s)
Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Vejiga Urinaria/metabolismo , Animales , Apamina/farmacología , Interpretación Estadística de Datos , Estimulación Eléctrica , Genes Reporteros/genética , Técnicas In Vitro , Ratones , Ratones Noqueados , Contracción Muscular/fisiología , Músculo Liso/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/efectos de los fármacos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Suramina/farmacología , Vejiga Urinaria/efectos de los fármacos , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
T-type calcium channels open in response to small depolarizations of the plasma membrane. The entry of two positive charges with every calcium ion leads to a further depolarization of the membrane, the low threshold spike, and opening of channels that have a higher threshold. In this manner, T-channels play an important pacemaker role in gating the activity of Na+ and Ca2+ channels. T-channels are preferentially expressed in dendrites, suggesting they play important roles in synaptic integration. Pharmacological evidence indicates that they are expressed in the receptive fields of sensory neurons, suggesting they play a primary role in nociception. Molecular cloning of the three T-channel genes has allowed detailed studies on their channel properties, pharmacology, distribution in the brain, up-regulation in animal models of disease, and provided the tools to screen for novel drugs. Studies on transgenic animals have provided the proof-of-concept that T-channels are important drug targets for the treatment of absence epilepsy and neuropathic pain. Mutations in ion channel genes, or channelopathies, have been found in many diseases. Similarly, T-channel gene mutations have been found in patients with childhood absence epilepsy. Considering the important role T-channels play in the thalamus, it is likely that T-channel mutations also contribute to a wider range of disorders characterized by thalamocortical dysrhythmia.
Asunto(s)
Encéfalo/metabolismo , Canales de Calcio Tipo T/metabolismo , Epilepsia/metabolismo , Dolor/metabolismo , Analgésicos/farmacología , Analgésicos/uso terapéutico , Anestésicos/farmacología , Anestésicos/uso terapéutico , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Encéfalo/efectos de los fármacos , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo T/química , Canales de Calcio Tipo T/efectos de los fármacos , Canales de Calcio Tipo T/genética , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Humanos , Mutación , Nociceptores/metabolismo , Dolor/tratamiento farmacológico , Dimensión del Dolor , Conformación ProteicaRESUMEN
Overactive bladder and incontinence are major medical issues, which lack effective therapy. Previously, we showed (Meredith AL, Thornloe KS, Werner ME, Nelson MT, and Aldrich RW. J Biol Chem 279: 36746-36752, 2004) that the gene mSlo1 encodes large-conductance Ca2+-activated K+ (BK) channels of urinary bladder smooth muscle (UBSM) and that ablation of mSlo1 leads to enhanced myogenic and nerve-mediated contractility and increased urination frequency. Here, we examine the in vivo urodynamic consequences and neurotransmitter dependence in the absence of the BK channel. The sensitivity of contractility to nerve stimulation was greatly enhanced in UBSM strips from Slo-/- mice. The stimulation frequency required to obtain a 50% maximal contraction was 8.3 +/- 0.9 and 19.1 +/- 1.8 Hz in Slo-/- and Slo+/+ mice, respectively. This enhancement is at least partially due to alterations in UBSM excitability, as muscarinic-induced Slo-/- contractility is elevated in the absence of neuronal activity. Muscarinic-induced Slo-/- contractility was mimicked by blocking BK channels with iberiotoxin (IBTX) in Slo+/+ strips, whereas IBTX had no effect on Slo-/- strips. IBTX also enhanced purinergic contractions of Slo+/+ UBSM but was without effect on purinergic contractions of Slo-/- strips. In vivo bladder pressure and urine output measurements (cystometry) were performed on conscious, freely moving mice. Slo-/- mice exhibited increased bladder pressures, pronounced pressure oscillations, and urine dripping. Our results indicate that the BK channel in UBSM has a very significant role in urinary function and dysfunction and as such likely represents an important therapeutic target.
Asunto(s)
Neurotransmisores/metabolismo , Canales de Potasio Calcio-Activados/genética , Canales de Potasio Calcio-Activados/metabolismo , Vejiga Urinaria/fisiología , Incontinencia Urinaria/fisiopatología , Urodinámica , Animales , Femenino , Eliminación de Gen , Canales de Potasio de Gran Conductancia Activados por el Calcio , Masculino , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/inervación , Músculo Liso/fisiología , Péptidos/farmacología , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Presión , Vejiga Urinaria/inervación , Incontinencia Urinaria/metabolismoRESUMEN
Spontaneous transient outward K(+) currents (STOCs) elicited by Ca(2+) sparks and steady-state K(+) currents modulate vascular reactivity, but effects of artery size, diabetic dyslipidemia, and exercise on these differentially regulated K(+) currents are unclear. We studied the conduit arteries and microvessels of male Yucatan swine assigned to one of three groups for 20 wk: control (C, n = 7), diabetic dyslipidemic (DD, n = 6), or treadmill-trained DD animals (DDX, n = 7). Circumflex artery blood flow velocity obtained with intracoronary Doppler and lumen diameters obtained by intravascular ultrasound enabled calculation of absolute coronary blood flow (CBF). Ca(2+) sparks were determined in pressurized microvessels, and perforated patch clamp assessed K(+) current in smooth muscle cells isolated from conduits and microvessels. Baseline CBF in DD was decreased versus C. In pressurized microvessels, Ca(2+) spark activity was significantly lower in DD versus C and DDX (P < 0.05 vs. DDX). STOCs were pronounced in microvessel (approximately 35 STOCs/min) in sharp contrast to conduit cells ( approximately 2 STOCs/min). STOCs were decreased by 86% in DD versus C and DDX in microvessels; in contrast, there was no difference in STOCs across groups in conduit cells. Steady-state K(+) current in microvessels was decreased in DD and DDX versus C; in contrast, steady-state K(+) current in conduit cells was decreased in DDX versus DD and C. We conclude that steady-state K(+) current and STOCs are differentially regulated in conduit versus microvessels in health and diabetic dyslipidemia. Exercise prevented diabetic dyslipidemia-induced decreases in baseline CBF, possibly via STOC-regulated basal microvascular tone.
Asunto(s)
Circulación Coronaria/fisiología , Angiopatías Diabéticas/fisiopatología , Hiperlipidemias/fisiopatología , Condicionamiento Físico Animal/fisiología , Canales de Potasio/fisiología , Animales , Señalización del Calcio/fisiología , Angiopatías Diabéticas/diagnóstico por imagen , Hiperlipidemias/diagnóstico por imagen , Masculino , Microcirculación/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Porcinos , Porcinos Enanos , UltrasonografíaRESUMEN
Inward currents in root cap protoplasts of the aluminum-tolerant cultivar, Dade, of Phaseolus vulgaris L. were investigated using the whole-cell patch-clamp technique. The properties of these currents were similar to those seen in inward rectifying K+ channels in other plant tissues. Replacing bath K+ with Na+ nearly abolished the observed currents. Higher bath K+ concentrations increased inward currents. AlCl3 in pH 4.7 bath solutions caused inward K+ currents to activate more rapidly and at more positive voltages when compared with AlCl3 free solutions. In 10 microM AlCl3 the activated inward K+ currents were significantly larger than in the AlCl3-free solution at all voltages except at the most negative voltage of -174 mV and the least negative of -74 mV. In contrast, in 80 microM Al3+, when hyperpolarizing voltages were most negative, the inward K+ currents were inhibited relative to the currents in 10 microM AlCl3. Enhancement of inward K+ currents by AlCl3 is consistent with Al3+ binding to the external surface of the root cap protoplast, decreasing the surface charge, thus causing the channels to sense a more negative membrane potential. Inhibition of inward K+ currents with higher AlCl3 concentrations and more negative voltages is consistent with Al3+ block of K+ channels.
Asunto(s)
Aluminio/farmacología , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Phaseolus/fisiología , Raíces de Plantas/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Protoplastos/fisiología , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Resistencia a Medicamentos/fisiología , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Phaseolus/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Protoplastos/efectos de los fármacos , Sodio/metabolismoRESUMEN
This commentary presents the proceedings of the symposium sponsored by Cardiovascular Section of American Physiological Society in San Diego, CA on 12 April 2003. The major focus of this symposium was on the actions and physiological relevance of several novel Ca2+ signalling mechanisms in vascular smooth muscle (VSM) cells. Five important topics were presented in this symposium including the discovery and roles of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) in mediating Ca2+ release, Ca2+ sparks and activation of plasma membrane KCa channels in VSM cells, the role of cADPR-mediated activation of ryanodine receptors in the control of vascular tone, the role of [Ca2+]i in mechanotransduction in the arterioles, and interactions of mitochondrial Ca2+ release and SR Ca2+ mobilization. The purpose of this symposium was to promote discussions and exchange of ideas between scientists with interests in Ca2+ signalling mechanisms and those with interests in vascular physiology and pharmacology. The cross-fertilization of ideas is expected to greatly advance our understanding of the physiological and pharmacological relevance of these new Ca2+ signalling mechanisms.
Asunto(s)
Señalización del Calcio/fisiología , Músculo Liso Vascular/metabolismo , Arteriolas/metabolismo , Transporte Biológico , Calcio/metabolismo , ADP-Ribosa Cíclica/metabolismo , Humanos , Integrinas/metabolismo , Mitocondrias/metabolismo , NADP/análogos & derivados , NADP/metabolismo , Canales de Potasio/metabolismoRESUMEN
1. The large-conductance calcium-activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore-forming alpha-subunit and an accessory, smooth muscle-specific, beta1-subunit. 2. Our aim was to determine the functional role of the beta1-subunit of the BK channel in controlling the contractions of UBSM by using BK channel beta1-subunit 'knock-out' (KO) mice. 3. The beta-galactosidase reporter (lacZ gene) was targeted to the beta1 locus, which provided the opportunity to examine the expression of the beta1-subunit in UBSM. Based on this approach, the beta1-subunit is highly expressed in UBSM. 4. BK channels lacking beta1-subunits have reduced activity, consistent with a shift in BK channel voltage/Ca2+ sensitivity. 5. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice. 6. The effects of the beta1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from beta1-subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips. 7. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from beta1-subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the beta1-subunit. 8. The results indicate that the beta1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder.
Asunto(s)
Contracción Muscular/fisiología , Músculo Liso/fisiología , Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Vejiga Urinaria/fisiología , Animales , Femenino , Técnicas In Vitro , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Masculino , Ratones , Ratones Noqueados/genética , Isoformas de Proteínas/fisiologíaRESUMEN
The goal of the present study was to test the hypothesis that local Ca(2+) release events (Ca(2+) sparks) deliver high local Ca(2+) concentration to activate nearby Ca(2+)-sensitive K(+) (BK) channels in the cell membrane of arterial smooth muscle cells. Ca(2+) sparks and BK channels were examined in isolated myocytes from rat cerebral arteries with laser scanning confocal microscopy and patch-clamp techniques. BK channels had an apparent dissociation constant for Ca(2+) of 19 microM and a Hill coefficient of 2.9 at -40 mV. At near-physiological intracellular Ca(2+) concentration ([Ca(2+)](i); 100 nM) and membrane potential (-40 mV), the open probability of a single BK channel was low (1.2 x 10(-6)). A Ca(2+) spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca(2+) spark, BK channel activity increases 6 x 10(5)-fold to 6 x 10(3)-fold, which corresponds to approximately 30 microM to 4 microM spark Ca(2+) concentration. 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester caused the disappearance of all Ca(2+) sparks while leaving the transient BK currents unchanged. Our results support the idea that Ca(2+) spark sites are in close proximity to the BK channels and that local [Ca(2+)](i) reaches micromolar levels to activate BK channels.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Músculo Liso Vascular/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Animales , Arterias Cerebrales , Quelantes/metabolismo , Ácido Egtácico/metabolismo , Femenino , Canales de Potasio de Gran Conductancia Activados por el Calcio , Masculino , Microscopía Confocal , Músculo Liso Vascular/citología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-DawleyRESUMEN
Elevated intracellular Ca(2+) ([Ca(2+)](i)) has been implicated in contractile and phenotypic changes in arterial smooth muscle during hypertension. This study examined the role of membrane potential and [Ca(2+)](i) in altered gene expression in cerebral arteries of a rat (Dahl) genetic model of salt-sensitive hypertension. Cerebral arteries from hypertensive animals (Dahl salt-sensitive) exhibited a tonic membrane depolarization of approximately 15 mV compared with normotensive (Dahl salt-resistant) animals. Consistent with this membrane depolarization, voltage-dependent K(+) currents were decreased in cerebral artery myocytes isolated from hypertensive animals. Arterial wall Ca(2+) was elevated in cerebral arteries from hypertensive animals, an effect reversed by diltiazem, a blocker of voltage-dependent Ca(2+) channels. This depolarization-induced increase in [Ca(2+)](i) was associated with increased activation of the transcription factor, cAMP response element binding protein, and increased expression of the immediate early gene c-fos, both of which are reversed by acute exposure to the voltage-dependent Ca(2+) channel blocker nisoldipine. This study provides the first information linking altered Ca(2+) handling to changes in gene expression in cerebral arteries during hypertension.
Asunto(s)
Calcio/metabolismo , Arterias Cerebrales/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Expresión Génica/fisiología , Potenciales de la Membrana/fisiología , Músculo Liso Vascular/fisiología , Nisoldipino/farmacología , Canales de Potasio con Entrada de Voltaje/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Ratas , Ratas Endogámicas DahlRESUMEN
Large-conductance, Ca(2+)-activated K(+) (BK(Ca)) channels in smooth muscle cells are unique because they integrate changes in both intracellular Ca(2+) and membrane potential. Protein kinases such as cAMP-dependent protein kinase, cGMP-dependent protein kinase and protein kinase C can affect tissue function by 'tuning' the apparent Ca(2+)- and/or voltage-sensitivity of the BK(Ca) channel to physiological changes in both Ca(2+) concentrations and membrane potential. However, despite the central importance of kinase-mediated modulation of BK(Ca) channels in different smooth muscle tissues, many key issues, including the sites and mechanisms of actions of protein kinases, remain unresolved. In this article, the role of protein kinases in the regulation of BK(Ca) channels is discussed.
Asunto(s)
Músculo Liso/enzimología , Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Proteínas Quinasas/fisiología , Animales , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio , Músculo Liso/metabolismo , Músculo Liso/fisiología , Fosforilación , Canales de Potasio/metabolismoRESUMEN
Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, and this inhibition is relieved by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. The role of PLB in regulating Ca(2+) release through ryanodine-sensitive Ca(2+) release channels, measured as Ca(2+) sparks, was examined using smooth muscle cells of cerebral arteries from PLB-deficient ("knockout") mice (PLB-KO). Ca(2+) sparks were monitored optically using the fluorescent Ca(2+) indicator fluo 3 or electrically by measuring transient large-conductance Ca(2+)-activated K(+) (BK) channel currents activated by Ca(2+) sparks. Basal Ca(2+) spark and transient BK current frequency were elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, an activator of adenylyl cyclase, increased the frequency of Ca(2+) sparks and transient BK currents in cerebral arteries from control mice. However, forskolin had little effect on the frequency of Ca(2+) sparks and transient BK currents from PLB-KO cerebral arteries. Forskolin or PLB-KO increased SR Ca(2+) load, as measured by caffeine-induced Ca(2+) transients. This study provides the first evidence that PLB is critical for frequency modulation of Ca(2+) sparks and associated BK currents by PKA in smooth muscle.
Asunto(s)
Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/fisiología , Calcio/fisiología , Arterias Cerebrales/fisiología , AMP Cíclico/fisiología , Músculo Liso Vascular/fisiología , Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Compuestos de Anilina , Animales , Cafeína/farmacología , Señalización del Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Arterias Cerebrales/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Canales de Potasio de Gran Conductancia Activados por el Calcio , Ratones , Ratones Noqueados , Modelos Biológicos , Músculo Liso Vascular/efectos de los fármacos , Rianodina/farmacología , Tionucleótidos/farmacología , XantenosRESUMEN
Activation of ATP-sensitive potassium (K(ATP)) channels can regulate smooth muscle function through membrane potential hyperpolarization. A critical issue in understanding the role of K(ATP) channels is the relationship between channel activation and the effect on tissue function. Here, we explored this relationship in urinary bladder smooth muscle (UBSM) from the detrusor by activating K(ATP) channels with the synthetic compounds N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (ZD-6169) and levcromakalim. The effects of ZD-6169 and levcromakalim on K(ATP) channel currents in isolated UBSM cells, on action potentials, and on related phasic contractions of isolated UBSM strips were examined. ZD-6169 and levcromakalim at 1.02 and 2.63 microM, respectively, caused half-maximal activation (K1/2) of K(ATP) currents in single UBSM cells (see Heppner TJ, Bonev A, Li JH, Kau ST, and Nelson MT. Pharmacology 53: 170-179, 1996). In contrast, much lower concentrations (K(1/2) = 47 nM for ZD-6169 and K1/2 = 38 nM for levcromakalim) caused inhibition of action potentials and phasic contractions of UBSM. The results suggest that activation of <1% of K(ATP) channels is sufficient to inhibit significantly action potentials and the related phasic contractions.
Asunto(s)
Amidas/farmacología , Benzofenonas/farmacología , Cromakalim/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/fisiología , Canales de Potasio/fisiología , Vejiga Urinaria/fisiología , Potenciales de Acción/fisiología , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/fisiología , Animales , Gliburida/farmacología , Guanosina Trifosfato/farmacología , Cobayas , Técnicas In Vitro , Músculo Liso/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacosRESUMEN
The transcription factor NFAT (nuclear factor of activated T-cells) plays a central role in mediating Ca(2+)-dependent gene transcription in a variety of cell types. Sustained increases in intracellular calcium concentration ([Ca(2+)]i) are presumed to be required for NFAT dephosphorylation by the Ca(2+)/calmodulin-dependent protein calcineurin and its subsequent nuclear translocation. Here, we provide the first identification and characterization of NFAT in native smooth muscle, showing that NFAT4 is the predominant isoform detected by reverse transcriptase-polymerase chain reaction and Western blot analysis. PDGF induces NFAT4 translocation in smooth muscle, leading to an increase in NFAT transcriptional activity. NFAT4 activation by PDGF depends on Ca(2+) entry through voltage-dependent Ca(2+) channels, because its nuclear accumulation is prevented by the Ca(2+) channel blocker nisoldipine and the K(+) channel opener pinacidil. Interestingly, elevation of [Ca(2+)]i by membrane depolarization or ionomycin treatment are not effective stimuli for NFAT4 nuclear accumulation, indicating that Ca(2+) influx is necessary but not sufficient for NFAT4 activation. In contrast, membrane depolarization readily activates the Ca(2+)-dependent transcription factor CREB (cAMP-responsive element-binding protein). The calcineurin blockers CsA and FK506 also prevented the PDGF-induced NFAT4 nuclear localization. These results indicate that both the nature of the calcium signal and PDGF-induced modulation of nuclear import-export of NFAT are critical for NFAT4 activation in this tissue.
Asunto(s)
Señalización del Calcio , Proteínas de Unión al ADN/metabolismo , Músculo Liso/metabolismo , Proteínas Nucleares , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/genética , Femenino , Ratones , Músculo Liso/efectos de los fármacos , Factores de Transcripción NFATC , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Large-conductance Ca(2+)-dependent K(+) (BK(Ca)) channels play a critical role in regulating urinary bladder smooth muscle (UBSM) excitability and contractility. Measurements of BK(Ca) currents and intracellular Ca(2+) revealed that BK(Ca) currents are activated by Ca(2+) release events (Ca(2+) sparks) from ryanodine receptors (RyRs) in the sarcoplasmic reticulum. The goals of this project were to characterize Ca(2+) sparks and BK(Ca) currents and to determine the voltage dependence of the coupling of RyRs (Ca(2+) sparks) to BK(Ca) channels in UBSM. Ca(2+) sparks in UBSM had properties similar to those described in arterial smooth muscle. Most Ca(2+) sparks caused BK(Ca) currents at all voltages tested, consistent with the BK(Ca) channels sensing approximately 10 microM Ca(2+). Membrane potential depolarization from -50 to -20 mV increased Ca(2+) spark and BK(Ca) current frequency threefold. However, membrane depolarization over this range had a differential effect on spark and current amplitude, with Ca(2+) spark amplitude increasing by only 30% and BK(Ca) current amplitude increasing 16-fold. A major component of the amplitude modulation of spark-activated BK(Ca) current was quantitatively explained by the known voltage dependence of the Ca(2+) sensitivity of BK(Ca) channels. We, therefore, propose that membrane potential, or any other agent that modulates the Ca(2+) sensitivity of BK(Ca) channels, profoundly alters the coupling strength of Ca(2+) sparks to BK(Ca) channels.