RESUMEN
BACKGROUND: Splenic vein ligation may result in sinistral (left-sided) portal hypertension and gastrointestinal haemorrhage. The aim of this study was to analyse the pathogenesis of sinistral portal hypertension following splenic vein ligation in pancreaticoduodenectomy. METHODS: Patients who underwent pancreaticoduodenectomy for pancreatic cancer between January 2005 and December 2012 were included in this retrospective study. The venous flow pattern from the spleen and splenic hypertrophy were examined after surgery. RESULTS: Of 103 patients who underwent pancreaticoduodenectomy with portal vein resection, 43 had splenic vein ligation. There were two predominant venous flow patterns from the spleen. In the varicose route (27 patients), flow from the spleen passed to colonic varices and/or other varicose veins. In the non-varicose route, flow from the spleen passed through a splenocolonic collateral (14 patients) or a spontaneous splenorenal shunt (2 patients). The varicose route was associated with significantly greater splenic hypertrophy than the non-varicose route (median splenic hypertrophy ratio 1·52 versus 0·94; P < 0·001). All patients with the varicose route had colonic varices, and none had a right colic marginal vein at the hepatic flexure. CONCLUSION: Pancreaticoduodenectomy with splenic vein ligation may lead to sinistral portal hypertension. To avoid the development of varices, it is important to preserve the right colic marginal vein. Reconstruction of the splenic vein should be considered if the right colic marginal vein is divided.
Asunto(s)
Hipertensión Portal/etiología , Neoplasias Pancreáticas/cirugía , Pancreaticoduodenectomía/efectos adversos , Vena Esplénica/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Circulación Colateral/fisiología , Várices Esofágicas y Gástricas/etiología , Várices Esofágicas y Gástricas/fisiopatología , Femenino , Humanos , Hipertensión Portal/fisiopatología , Ligadura/efectos adversos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/fisiopatología , Estudios Retrospectivos , Bazo/irrigación sanguínea , Esplenomegalia/etiología , Esplenomegalia/fisiopatologíaRESUMEN
AIMS: A next-generation, Illumina-based sequencing approach was used to characterize the bacterial community at ten sites along the Upper Mississippi River to evaluate shifts in the community potentially resulting from upstream inputs and land use changes. Furthermore, methodological parameters including filter size, sample volume and sample reproducibility were evaluated to determine the best sampling practices for community characterization. METHODS AND RESULTS: Community structure and diversity in the river was determined using Illumina next-generation sequencing technology and the V6 hypervariable region of 16S rDNA. A total of 16,400 operational taxonomic units (OTUs) were observed (4594 ± 824 OTUs per sample). Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and Verrucomicrobia accounted for 93.6 ± 1.3% of all sequence reads, and 90.5 ± 2.5% belonged to OTUs shared among all sites (n = 552). Among nonshared sequence reads at each site, 33-49% were associated with potentially anthropogenic impacts upstream of the second sampling site. Alpha diversity decreased with distance from the pristine headwaters, while rainfall and pH were positively correlated with diversity. Replication and smaller filter pore sizes minimally influenced the characterization of community structure. CONCLUSIONS: Shifts in community structure are related to changes in the relative abundance, rather than presence/absence of OTUs, suggesting a 'core bacterial community' is present throughout the Upper Mississippi River. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is among the first to characterize a large riverine bacterial community using a next-generation-sequencing approach and demonstrates that upstream influences and potentially anthropogenic impacts can influence the presence and relative abundance of OTUs downstream resulting in significant variation in community structure.
Asunto(s)
Bacterias/clasificación , Biodiversidad , Ríos/microbiología , Actinobacteria/clasificación , Actinobacteria/genética , Bacterias/genética , Bacteroidetes/clasificación , Bacteroidetes/genética , ADN Bacteriano/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Concentración de Iones de Hidrógeno , Minnesota , Proteobacteria/clasificación , Proteobacteria/genética , ARN Ribosómico 16S/genética , Lluvia , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
BACKGROUND AND PURPOSE: The functional roles of M(2) and M(3) muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve-induced contractions in the ileum using mutant mice lacking M(2) or M(3) receptor subtypes. EXPERIMENTAL APPROACH: Contractile responses to transmural electrical (TE) stimulation were isometrically recorded in ileal segments from M(2)-knockout (KO), M(3)-KO, M(2)/M(3)-double KO, and wild-type mice. KEY RESULTS: TE stimulation at 2-50 Hz frequency-dependently evoked a fast, brief contraction followed by a slower, longer one in wild-type, M(2)-KO or M(3)-KO mouse preparations. Tetrodotoxin blocked both the initial and later contractions, while atropine only inhibited the initial contractions. The initial cholinergic contractions were significantly greater in wild-type than M(2)-KO or M(3)-KO mice; the respective mean amplitudes at 50 Hz were 91, 74 and 68 % of 70mM K(+)-induced contraction. Pretreatment with pertussis toxin blocked the cholinergic contractions in M(3)-KO but not in M(2)-KO mice. Cholinergic contractions also remained in wild-type preparations, but their sizes were reduced by 20-30 % at 10-50 Hz. In M(2)/M(3)-double KO mice, TE stimulation evoked only slow, noncholinergic contractions, which were significantly greater in sizes than in any of the other three mouse strains. CONCLUSION AND IMPLICATIONS: These results demonstrate that M(2) and M(3) receptors participate in mediating cholinergic contractions in mouse ileum with the latter receptors assuming a greater role. Our data also suggest that the lack of both M(2) and M(3) receptors causes upregulation of noncholinergic excitatory innervation of the gut smooth muscle.
Asunto(s)
Músculo Liso/fisiología , Sistema Nervioso Parasimpático/fisiología , Receptor Muscarínico M2/fisiología , Receptor Muscarínico M3/fisiología , Anestésicos Locales/farmacología , Animales , Atropina/farmacología , Capsaicina/farmacología , Estimulación Eléctrica , Femenino , Íleon/efectos de los fármacos , Contracción Isométrica/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Antagonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Sistema Nervioso Parasimpático/efectos de los fármacos , Toxina del Pertussis/farmacología , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/efectos de los fármacos , Receptor Muscarínico M3/genética , Tetrodotoxina/farmacologíaRESUMEN
1 The muscarinic receptor plays a key role in the parasympathetic nervous control of various peripheral tissues including gastrointestinal tract. The neurotransmitter acetylcholine, via activating muscarinic receptors that exist in smooth muscle, produces its contraction. 2 There is the opening of cationic channels as an underlying mechanism. The opening of cationic channels results in influxes of Ca2+ via the channels into the cell and also via voltage-dependent Ca2+ channels which secondarily opened in response to the depolarization, providing an amount of Ca2+ for activation of the contractile proteins. 3 Electrophysiological and pharmacological studies have shown that the cationic channels as well as muscarinic receptors exist in many visceral smooth muscle cells. However, the activation mechanisms of the cationic channels are still unclear. 4 In this article, we summarize the current knowledge of the muscarinic receptor-operated cationic channels, focusing on the receptor subtype, G protein and other signalling molecules that are involved in activation of these channels and on the molecular characteristics of the channel. This will improve strategies aimed at developing new selective pharmacological agents and understanding the activation mechanism and functions of these channels in physiological systems.
Asunto(s)
Señalización del Calcio , Tracto Gastrointestinal/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/metabolismo , Animales , Proteínas de Unión al GTP/metabolismo , Tracto Gastrointestinal/inervación , Humanos , Activación del Canal Iónico , Potenciales de la Membrana , Músculo Liso/inervación , Quinasa de Cadena Ligera de Miosina/metabolismo , Sistema Nervioso Parasimpático/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Canales Catiónicos TRPC/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Regulation of vasoactive intestinal peptide (VIP) release by nitric oxide (NO) was investigated in the hamster jejunum. Electrical field stimulation and applied NO (3-100 microM) evoked biphasic hyperpolarizations consisting of an initial transient hyperpolarizing component followed by a second more slowly developing component (late component). The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (200 microM) abolished the biphasic inhibitory junction potential evoked by electrical field stimulation. The NO scavenger oxyhemoglobin (50 microM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 10 microM) abolished both components of the inhibitory junction potentials and the NO-induced hyperpolarizations. VIP(6-28) (1 microM), which abolished VIP (3 microM)-induced hyperpolarizations, also inhibited the late components of the inhibitory junction potentials and the NO-induced hyperpolarizations. ODQ inhibited VIP release and cAMP production by electrical field stimulation and NO application. N(6)-2,0-Dibutyryladenosine 3',5'-cyclic monophosphate (0.1-3 mM) caused a membrane hyperpolarization. These results suggest that NO may stimulate VIP release from enteric nerves in the hamster jejunum. In addition, we propose that NO and NO-stimulated VIP contribute to the early and late components of the inhibitory junction potentials, respectively, in the circular smooth muscle cells of the hamster jejunum.
Asunto(s)
GMP Cíclico/análogos & derivados , Yeyuno/inervación , Músculo Liso/inervación , Plexo Mientérico/metabolismo , Inhibición Neural/fisiología , Neuronas Nitrérgicas/metabolismo , Óxido Nítrico/metabolismo , Transmisión Sináptica/fisiología , Péptido Intestinal Vasoactivo/metabolismo , Animales , Cricetinae , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Yeyuno/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mesocricetus , Músculo Liso/metabolismo , Plexo Mientérico/citología , Plexo Mientérico/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Inhibición Neural/efectos de los fármacos , Neuronas Nitrérgicas/citología , Neuronas Nitrérgicas/efectos de los fármacos , Óxido Nítrico/farmacología , Oxadiazoles/farmacología , Oxihemoglobinas/metabolismo , Oxihemoglobinas/farmacología , Fragmentos de Péptidos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Purinonas/farmacología , Rolipram/farmacología , Transmisión Sináptica/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
1. Effects of agents, which affect microtubule polymerization-depolymerization cycle, on Ba2+ current (IBa) flowing through voltage-gated Ca2+ channels and carbachol (CCh)-induced sustained suppression of IBa were examined in whole-cell voltage-clamped smooth muscle cells of guinea-pig ileum. 2. offchicine (100 microM) and vinblastine (100 microM), microtubule depolymerizers, increased the ampitude of IBa. Lumicolchicine (100 microM), an inactive analogue of colchicine, had no effect on IBa. 3. Taxol (1 - 100 microM), a microtubule polymerizer, decreased IBa in a concentration-dependent manner and accelerated the rate of inactivation of IBa. Baccatin III (100 microM), an inactive analogue of taxol, had no effect on IBa. 4. Colchicine (100 microM) and vinblastine (100 microM), but not lumicolchicine (100 microM), decreased or abolished the sustained component of CCh (10 microM)-induced IBa suppression. 5. Pretreatment with taxol (10 - 100 microM) resulted in a concentration-dependent decrease in IBa and the action of CCh on IBa. The inhibitory effects of taxol and CCh on IBa were not additive. 6. Colchicine (100 microM) or taxol (100 microM) had no effect on voltage-gated K+ channel current or CCh-induced non-selective cationic channel current. 7. These results suggest that polymerization of microtubules leads to suppression of Ca2+ channel activity, and that muscarinic sustained suppression of Ca2+ channel current is mediated by a signal transduction element which involves microtubule cytoskeleton.
Asunto(s)
Canales de Calcio Tipo N , Canales de Calcio/fisiología , Citoesqueleto/efectos de los fármacos , Íleon/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Receptores Muscarínicos/fisiología , Animales , Bario/farmacología , Canales de Calcio/efectos de los fármacos , Carbacol/farmacología , Membrana Celular/efectos de los fármacos , Colchicina/farmacología , Citocalasina B/farmacología , Cobayas , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Paclitaxel/farmacología , Técnicas de Placa-Clamp , Faloidina/farmacología , Receptores Muscarínicos/efectos de los fármacos , Vinblastina/farmacologíaRESUMEN
1. Non-adrenergic, non-cholinergic (NANC) inhibitory transmission in chicken gizzard was studied by use of intracellular microelectrode techniques. Changes in membrane potential in response to NANC nerve stimulation were recorded in the gizzard smooth muscle pretreated with atropine (1 microM) and guanethidine (1 microM). 2. Field stimulation of the intramural nerves (FS) evoked inhibitory junction potentials (i.j.ps) which were abolished by tetrodotoxin (1 microM), but not inhibited at all by K+ channel blockers including apamin (0.5 microM), tetraethylammonium (TEA, 10 mM), charybdotoxin (0.2 microM) and glibenclamide (10 microM). 3. NG-nitro-L-arginine (3 mM), an inhibitor of nitric oxide (NO) synthase, inhibited i.j.ps. The effect was reversed by L-arginine (3 mM), but not by D-arginine (3 mM). 4. 8-Bromo cyclic GMP (100 microM), a membrane permeable analogue of cyclic GMP, produced a membrane hyperpolarization which was blocked by TEA (10 mM) or glibenclamide (10 microM). 5. NO at concentrations of up to 400 microM affected neither i.j.ps nor resting membrane potential. On the other hand, NO (80 microM) caused the membrane to hyperpolarize in the smooth muscle of guinea-pig ileum. 6. These results suggest that in the chicken gizzard, NANC i.j.ps may not arise from opening of conventional types of K+ channel and that NO seems unlikely to be involved in the generation of i.j.ps. A possible mechanism by which the inhibitory effect of NG-nitro-L-arginine on i.j.ps was brought about will be discussed.
Asunto(s)
Molleja de las Aves/inervación , Transmisión Sináptica , Animales , Apamina/farmacología , Pollos , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Óxido Nítrico/farmacología , Nitroarginina/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio/fisiología , Compuestos de Tetraetilamonio/farmacologíaRESUMEN
1. To see if arachidonic acid (AA) plays a role in the sustained suppression of voltage-gated calcium channel currents produced by muscarinic receptor stimulation by carbachol (CCh), the effects of AA on membrane currents were examined in whole-cell voltage-clamped smooth muscle cells of the guinea-pig ileum. 2. In cells bathed in Ba2+ PSS and dialysed with Cs(+)-based low EGTA (0.05 mM) pipette solution, and in which Ba2+ current (IBa) flowing through voltage-gated calcium channels was evoked repeatedly by stepping to 0 mV from the holding potential of -60 mV, AA (1-30 microM), applied extracellularly, gradually suppressed IBa in a concentration-dependent manner. The IBa suppression was observed even with 20 mM EGTA in the pipette. 3. AA (3 microM) and CCh (10 microM) shifted the voltage-dependent inactivation curve of IBa in the negative potential direction, but the effect of AA differed from that of CCh in that an accompanying appreciable decrease in the slope was observed. 4. The sustained suppression of IBa induced by CCh (10 microM) remained almost unaltered after pretreatment with 4-bromophenacyl bromide (10 microM), an inhibitor of phospholipase A2, or a combination of indomethacin (10 microM), an inhibitor of the cyclo-oxygenase pathway, and nordihydroguaiaretic acid (10 microM), an inhibitor of the lipoxygenase pathway. 5. In cells bathed in Ca2+ PSS and dialysed with K(+)-based pCa 6.5 pipette solution, voltage-dependent Ca2+ current (ICa) and K+ current (IK) were recorded simultaneously. AA (3 microM) suppressed IK as well as ICa, whereas CCh (10 microM) suppressed ICa but not IK. 6. We conclude from these results that AA or its metabolite is unlikely to be involved in the sustained suppression of voltage-gated calcium channel current induced by muscarinic receptor stimulation in guinea-pig ileal smooth muscle cells.
Asunto(s)
Ácido Araquidónico/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Activación del Canal Iónico/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Receptores Muscarínicos/fisiología , Animales , Ácido Araquidónico/metabolismo , Bario/metabolismo , Bario/farmacología , Calcio/metabolismo , Calcio/farmacología , Carbacol/farmacología , Células Cultivadas , Inhibidores de la Ciclooxigenasa/farmacología , Depresión Química , Cobayas , Íleon/efectos de los fármacos , Íleon/fisiología , Agonistas Muscarínicos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Receptores Muscarínicos/efectos de los fármacosRESUMEN
1. To characterize increases in cytosolic free Ca2+ concentration ([Ca2+]i) associated with discharge of action potentials, membrane potential and [Ca2+]i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques. 2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca2+]i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca2+]i transients with an almost constant amplitude. 3. Nicardipine (1 microM) and La3+ (10 microM), blockers of voltage-dependent Ca2+ channels (VDCCs), abolished both the action potential discharge and the [Ca2+]i transient. 4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca2+-activated K+ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca2+]i transients. 5. Thapsigargin (1 microM), an inhibitor of SR Ca2+-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca2+]i transients without affecting action potentials. In addition. in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca2+ current but exerted a similar effect on its concomitant rise in [Ca2+]i to that on the action potential-triggered [Ca2+]i transient. 6. Similar action potential-triggered [Ca2+]i transients were induced by brief exposures to high-K+ solution. They were not decreased, but rather increased, after depletion of intracellular Ca2+ stores by a combination of ryanodine (30 microM) and caffeine (10 mM) through an open-lock of Ca2+-induced Ca2+ release (CICR)-related channels. 7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca2+]i. Intracellular Ca2+ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca2+]i transients by replenishing cytosolic Ca2+. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca2+]i transient.
Asunto(s)
Potenciales de Acción/fisiología , Calcio/metabolismo , Íleon/metabolismo , Músculo Liso/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Cafeína/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Caribdotoxina/farmacología , Citosol/metabolismo , Fluorometría , Fura-2 , Cobayas , Íleon/fisiología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Músculo Liso/fisiología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Rianodina/farmacología , Tapsigargina/farmacologíaRESUMEN
1. Oscillations of cytosolic Ca2+ concentration ([Ca2+]i) evoked by carbachol (CCh; 2 microM), a muscarinic agonist, were detected as oscillatory changes of muscarinic receptor-coupled cationic current (Icat) in guinea-pig ileal smooth muscle cells by the whole cell patch-clamp technique. 2. Reduction of extracellular Ca2+ from 2 mM to 0.2 or 0.05 mM, during CCh-induced Icat oscillations, caused them to disappear or to decrease markedly in frequency. A return to 2 mM Ca2+ concentration restored the initial Icat oscillations. 3. Application of nifedipine (1-3 microM) or D600 (2-5 microM) to block the voltage-gated Ca2+ channel (VGCC) decreased the frequency of the ongoing Icat oscillations in the cells held at -20 mV, but it was without effect in cells held at -60 mV. 4. Displacement of the holding potential of -20 mV to -60 mV to deactivate VGCC produced a decrease, an increase or no noticeable change in the frequency of the Icat oscillations in different cells. Displacement to 20 mV to inactivate VGCC invariably produced a decrease in the frequency. In nifedipine-treated cells, the Icat oscillations varied in frequency voltage-dependently in a reverse and linear way within the range -80 to 40 mV. 5. Application of thapsigargin (1 or 2 microM), an inhibitor of Ca(2+)-ATPase in the membrane of internal Ca2+ stores, caused CCh-induced Icat oscillations to disappear with a progressing phase during which their amplitude, but not frequency, declined. 6. The results suggest that membrane Ca2+ entry has a crucial role to play in regulation of the frequency of CCh-induced [Ca2+]i oscillations in addition to persistence of their generation, and that the effect is brought about by a potential mechanism independent of Ca2+ store replenishment. They also provide evidence that two types of Ca2+ permeant channels, VGCC and an as yet unidentified channel, are involved in the Ca2+ entry responsible for modulation of [Ca2+]i oscillations.
Asunto(s)
Calcio/metabolismo , Carbacol/farmacología , Agonistas Muscarínicos/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Animales , Calcio/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiología , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Cobayas , Íleon/efectos de los fármacos , Íleon/metabolismo , Íleon/fisiología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso/fisiología , Tapsigargina/farmacologíaRESUMEN
1. The effect on membrane currents of infection of mouse neuroblastoma NA cells with rabies virus was studied by using the whole-cell patch clamp technique. 2. Three types of membrane currents, namely voltage-dependent Na+ current (I(Na)), delayed rectifier K+ current (I(K-DR)) and inward rectifier K+ current (I(K-IR)) were elicited in uninfected cells. 3. In cells 3 days after infection with the virus, no detectable change was observed in morphology and membrane capacitance, but I(Na) and I(K-IR) were significantly decreased in amplitude without any appreciable difference in the time course of current activation and inactivation. The voltage-dependence of I(Na) activation was significantly shifted in the positive direction along the voltage axis with a decreased slope. I(K-DR) remained almost unaltered after the viral infection. 4. The resting membrane potential, measured with a physiological K+ gradient across the cell membrane, was decreased (depolarized) after the viral infection. The depolarization was associated with the decreased amplitude of I(K-IR). 5. These results suggest that infection of mouse neuroblastoma NA cells with rabies virus causes reduction of functional expression of ion channels responsible for I(Na) and I(K-IR), and provide evidence for possible involvement of the change in membrane properties in the pathogenesis of rabies disease.
Asunto(s)
Neuroblastoma/fisiopatología , Neuroblastoma/virología , Virus de la Rabia , Rabia/fisiopatología , Animales , Membrana Celular/fisiología , Cricetinae , Impedancia Eléctrica , Potenciales de la Membrana/fisiología , Ratones , Canales de Potasio/fisiología , Canales de Sodio/fisiología , Células Tumorales CultivadasRESUMEN
1. Whole-cell patch-clamp recordings were made from smooth muscle cells isolated from the longitudinal muscle layer of guinea-pig ileum. Carbachol (acting at muscarinic receptors) or histamine (acting at H1 histamine receptors) suppressed Ca2+ channel current. The effect of either agonist had an initial transient component followed by a sustained component. 2. Wortmannin inhibited transient and sustained components of carbachol-induced Ca2+ channel current suppression: half-effective inhibitory concentrations (IC50) were 1.1 microM and 0.6 microM for the two components respectively. Wortmannin also inhibited the transient phase of carbachol-induced cationic current (IC50 1.6 microM) and Ca2+-dependent K+-current (IC50 1.7 microM). Wortmannin did not appear to produce any direct block of cationic channels or Ca2+ channels. 3. Intracellular application of the phospholipase inhibitor D609 (tricyclodecan-9-ylxanthogenate) inhibited transient and sustained components of histamine action on the Ca2+ channel current: the IC50 was about 130 microM for both components. Carbachol action on Ca2+ channels was also inhibited by D609. D609 had no significant direct blocking effect on Ca2+ channels, cationic channels activated by carbachol, or Ca2+-activated K+-current in response to flash-photolysis of caged-inositol 1,4,5-trisphosphate. 4. Micromolar concentrations of wortmannin and D609 are inhibitors of both components of spasmogen-induced Ca2+ channel suppression. The data suggest that both components are mediated by a common, or similar, signal transduction element which is a phospholipase C (PLC) or phospholipase D (PLD) isoform.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Carbacol/farmacología , Histamina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Cobayas , Intestino Delgado/efectos de los fármacos , Masculino , Receptores Histamínicos/efectos de los fármacos , Receptores Muscarínicos/efectos de los fármacos , Factores de TiempoRESUMEN
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
1 In guinea-pig ileal longitudinal muscle, muscarinic partial agonists, 4-(N-[3-chlorophenyl]-carbomoyloxy)-2-butynyl-trimethylammonium (McN-A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca(2+) concentration ([Ca(2+)]c) with a higher EC(50) than that of the full agonist carbachol. The maximum response of [Ca(2+)]c or tension was not much different among the three agonists. The Ca(2+) channel blocker nicardipine markedly inhibited the effects of all three agonists 2 The contractile response to any agonist was antagonized in a competitive manner by M(2) receptor selective antagonists (N,N'-bis[6-[[(2-methoyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride and 11-[[2-[(diethlamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one), and the apparent order of M(2) antagonist sensitivity was McN-A343>pilocarpine>carbachol. M(3) receptor selective antagonists, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN-A343, while darifenacin had a similar action in the case of pilocarpine. Both M(3) antagonists behaved in a competitive manner in the case of the carbachol response. 3 McN-A343 failed to release Ca(2+) from the intracellular stores, and the Ca(2+)-releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca(2+) sensitivity of the contractile proteins. 4 McN-A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. 5 The results suggest that muscarinic agonist-evoked contractions result primarily from the integration of Ca(2+) entry associated with the increased spike discharge and myofilaments Ca(2+) sensitization, and that Ca(2+) store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M(2) and M(3) receptors plays a crucial role in mediating the contraction response.
Asunto(s)
Íleon/fisiología , Agonistas Muscarínicos/farmacología , Músculo Liso/fisiología , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M3/agonistas , Transducción de Señal/fisiología , Cloruro de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamonio/farmacología , Animales , Calcio/metabolismo , Carbacol/farmacología , Cobayas , Íleon/metabolismo , Técnicas In Vitro , Masculino , Potenciales de la Membrana/fisiología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Pilocarpina/farmacología , Receptor Muscarínico M2/fisiología , Receptor Muscarínico M3/fisiologíaRESUMEN
(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
The Lombard reflex occurs when a speaker increases his vocal effort while speaking in the presence of ambient noise. The purpose of this study was to clarify whether the Lombard reflex can be evoked during controlled vocalization in an animal model. In decerebrate cats, repetitive electrical stimulation was applied to the midbrain periaqueductal gray (PAG) to evoke vocalization. Pure tone auditory stimulation was delivered through a loudspeaker. The activities of the laryngeal adductor muscle, diaphragm and external oblique abdominal muscle and the voice intensity were measured during PAG stimulation, in the presence and absence of the auditory stimulation. To clarify the effects of the auditory laryngeal reflex on the activity of laryngeal adductor motoneurons, the amplitude of the laryngeal reflex evoked by single shock stimulation of the superior laryngeal nerve was also measured during respiration, in the presence and absence of auditory stimulation. The sound made by the cats due to PAG-induced vocalization was augmented by exposure to auditory stimulation, and the activities of the laryngeal adductor muscle and external oblique abdominal muscle were also augmented. During respiration, auditory stimulation also increased the amplitude of the laryngeal reflex evoked in the laryngeal adductor muscle. These results demonstrate that the essential neuronal mechanisms for evoking the Lombard reflex exist within the brainstem.
Asunto(s)
Músculos Laríngeos/fisiología , Sustancia Gris Periacueductal/fisiología , Reflejo/fisiología , Vocalización Animal/fisiología , Estimulación Acústica , Animales , Gatos , Estado de Descerebración , Estimulación Eléctrica , Femenino , Masculino , RuidoRESUMEN
The neuronal mechanisms of sneezing were examined in precollicular-postmammillary decerebrate cats. Mechanical stimulation (frequency 20 Hz, peak-to-peak displacement 0.5 mm) of the nasal membrane evoked a series of sneezes. In the same preparation, electrical stimulation (duration 0.2 ms, frequency 10 Hz, 5-50 microA) delivered to the lower brainstem also evoked sneezes. The changes in EMG activity and subglottic pressure during brainstem-induced sneeze were similar to those recorded nasal-induced sneeze. The sneeze-evoking region was located along the ventromedial part of the spinal trigeminal nucleus and the adjacent pontine-medullary lateral reticular formation, and extended rostrocaudally from P4.0 to P14.5 according to the Horsley-Clarke coordinates. These results suggest that the sneeze-evoking region is a distinct structure of the brainstem, having a homogeneous function. This region appears to control the epipharyngeal, intrinsic laryngeal and respiratory muscles. The integrated activity of these muscles underlies the generation of a sneeze.
Asunto(s)
Tronco Encefálico/fisiología , Estornudo/fisiología , Músculos Abdominales/fisiología , Animales , Gatos , Diafragma/fisiología , Estimulación Eléctrica , Presión Intracraneal , Estimulación FísicaRESUMEN
Neurotensin reduced substance P-induced tension in ileal muscle strips and the relaxant effect was inhibited by a nonpeptide antagonist, SR 48692, 2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)car bonylamino]tricyclo(3.3.1.1.(3.7)decan-2-carboxylic acid with a half-maximal concentration (IC50) of 7.4+/-2.1 nM (n = 9) and a dissociation constant (Kb) of 0.9+/-0.2 nM. Neurotensin produced a contractile response in ileal muscle strips pretreated with apamin (50 nM) and in isolated chick rectums and both contractile effects were inhibited by SR 48692 with IC50 of 31.6+/-9.5 nM and Kh of 3.2+/-0.9 nM (n = 6) and with IC50 of 28.9+/-6.9 nM and Kb of 5.4+/-1.0 nM (n = 7), respectively. The Kb values for the contractile effects were not significantly different from each other, but significantly different from that for the relaxant effect, suggesting that both types of effect are mediated via distinct subtypes of neurotensin receptor in the intestinal smooth muscles. Contractile responses and excitatory junction potentials evoked by electrical stimulation of nonadrenergic, noncholinergic (NANC) nerves in isolated chick rectums were not inhibited by SR 48692 (up to 3.3 microM). This does not provide functional evidence for the idea that neurotensin acts as an unidentified excitatory neurotransmitter of NANC nerves in the avian rectum.
Asunto(s)
Intestinos/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Pirazoles/farmacología , Quinolinas/farmacología , Receptores de Neurotensina/antagonistas & inhibidores , Animales , Pollos , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Cobayas , Íleon/efectos de los fármacos , Íleon/fisiología , Técnicas In Vitro , Intestinos/fisiología , Masculino , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso/fisiología , Neurotensina/farmacología , Recto/efectos de los fármacos , Recto/inervación , Recto/fisiologíaRESUMEN
To characterize intracellular Ca2+ stores, the Ca(2+)-releasing effects of caffeine, carbachol and inositol 1,4,5-trisphosphate (IP3) were compared by measuring the drug-induced tension development in beta-escin-skinned longitudinal smooth muscle of guinea-pig ileum. Caffeine (20 mM), carbachol (10 or 100 microM) or IP3 (40 microM), applied after loading Ca2+ within intracellular stores, produced a transient rise in tension in a Ca(2+)-free solution. This change in tension occurred in response to release of Ca2+ from the stores. The effect of either caffeine or carbachol was markedly reduced or abolished after preceding application of the other drug. IP3 was without effect when applied subsequently to caffeine. The effects of carbachol and IP3 were abolished after combined treatment with ryanodine (30 microM) and caffeine (20 mM) which causes functional removal of caffeine-releasable Ca2+ stores, but not after combined treatment with ryanodine (30 microM) and carbachol (10 microM). The results suggest that caffeine, carbachol and IP3 all act on common Ca2+ stores to release Ca2+.
Asunto(s)
Cafeína/farmacología , Calcio/metabolismo , Carbacol/farmacología , Músculo Liso/efectos de los fármacos , Animales , Escina/farmacología , Cobayas , Íleon/efectos de los fármacos , Íleon/metabolismo , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Músculo Liso/metabolismo , Rianodina/farmacologíaRESUMEN
In guinea-pig dorsal root ganglion cells held under voltage-clamp at -80 mV, neurotensin elicited an inward current (I(NT)) whose amplitude increased with increasing neurotensin concentration (40-4000 nM). The effect was blocked by a nonpeptide neurotensin antagonist. I(NT) occurred in the absence of the extracellular Na(+), but not in the absence of the intracellular Cl(-), and it was outward directed by reversing the driving force for Cl(-). I(NT), like the gamma-amino-butyric acid (GABA)-induced Cl(-) current (I(GABA)), remained little changed after virtual elimination of cytosolic free-ionized Ca(2+) or after treatment with a Ca(2+)-activated Cl(-) channel blocker, but, in contrast to I(GABA) it was resistant to the I(GABA) blocker picrotoxin, slower in time course and more easily desensitized when repeatedly elicited. I(NT) and I(GABA) were additive to each other. AG-protein inhibitor markedly reduced I(NT), and a G-protein activator produced an inward current during which no current could be elicited by neurotensin. These results show that neurotensin exerts an effect to activate Ca(2+)-insensitive Cl(-) channels distinct from those activated by GABA in guinea-pig dorsal root ganglion cells, and the effect may arise through a G-protein-dependent mechanism.