RESUMO
Ion-transport properties of the epithelium of the cecum, the biggest fermental chamber in non-ruminant species, are largely unknown. Recently, in Ussing chamber experiments, segmental differences in basal short-circuit current (Isc) in rat corpus ceci were observed. The oral segment usually exhibited a much lower or even negative basal Isc in comparison with the aboral segment. The aim of the present study was the closer characterization of these differences. Basal Isc was inhibited by bumetanide and tetrodotoxin in both segments, whereas indomethacin reduced basal Isc only in the aboral corpus. Amiloride did not inhibit basal Isc suggesting that spontaneous anion secretion (but not electrogenic Na+ absorption via ENaC) contributes to the baseline current. In both segments, mucosally applied K+ channel blockers increased Isc indicating a spontaneous K+ secretion. Basolateral depolarization was used to characterize the ion conductances in the apical membrane. When a Cl- gradient was applied, apical Cl- conductance stimulated by carbachol and by forskolin was revealed. When the Cl- gradient was omitted and instead a K+ gradient was used to drive currents across apical K+ channels, a Ba2+-sensititve K+ conductance was observed in both segments, and carbachol stimulated this conductance leading to a negative Isc. Conversely, forskolin induced a positive Isc under these conditions which was dependent on the presence of mucosal Na+ consistent with electrogenic Na+ absorption. This current was reduced by amiloride and several blockers of members of the TRP channel superfamily. These results indicate that similar transport mechanisms are involved in electrogenic ion transport across cecal oral and aboral segments, but with a higher spontaneous prostaglandin production in the aboral segment responsible for higher basal transport rates of both anions and cations.
Assuntos
Ceco/metabolismo , Transporte de Íons/fisiologia , Íons/metabolismo , Amilorida/farmacologia , Animais , Bumetanida/farmacologia , Ceco/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cloretos/metabolismo , Colforsina/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Transporte de Íons/efeitos dos fármacos , Masculino , Potássio/metabolismo , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Ratos , Ratos Wistar , Sódio/metabolismo , Tetrodotoxina/farmacologiaRESUMO
Colloidal gold nanoparticles with a functionalized ligand shell were synthesized and used as new histamine receptor agonists. Mercaptoundecanoic acid moieties were attached to the surface of the nanoparticles and derivatized with native histamine. The multivalent presentation of the immobilized ligands carried by the gold nanoparticles resulted in extremely low activation concentrations for histamine receptors on rat colonic epithelium. As a functional read-out system, chloride secretion resulting from stimulation of neuronal and epithelial histamine H1 and H2 receptors was measured in Ussing chamber experiments. These responses were strictly attributed to the histamine entities as histamine-free particles Au-MUDOLS or the monovalent ligand AcS-MUDA-HA proved to be ineffective. The vitality of the tissues used was not impaired by the nanoparticles.
Assuntos
Ouro/química , Agonistas dos Receptores Histamínicos/farmacologia , Histamina/farmacologia , Nanopartículas Metálicas/química , Receptores Histamínicos/metabolismo , Animais , Histamina/química , Agonistas dos Receptores Histamínicos/química , Masculino , Ratos , Ratos WistarRESUMO
OBJECTIVES: How gaseous signalling molecules affect ion transport processes contributing to the physiological functions of the gastrointestinal tract under hypoxic conditions still needs to be clarified. The objective of the present study was to characterize the impact of gaseous signalling molecules on parameters of colonic ion transport during a hypoxia/reoxygenation cycle and the remaining secretory capacity of the epithelium after such a cycle. METHODS: Short-circuit current (Isc) and tissue conductance (Gt) recordings in Ussing chamber experiments were performed on rat colon samples using CORM-2 (putative CO donor; 35 and 350⯵M), sodium nitroprusside (NO donor; 100⯵M), NaHS (fast H2S donor; 10 - 1,000⯵M), GYY 4137 (slow H2S donor; 50⯵M) and Angeli's salt (HNO donor; 100⯵M) as donors for gasotransmitters. Inhibition of endogenous synthesis of H2S was operated by inhibitors of cystathionin-γ-lyase, i.e. dl-propargylglycine (1â¯mM) or ß-cyano-l-alanine (5â¯mM), and the inhibitor of cystathionine-ß-synthase, amino-oxyacetate (5â¯mM). RESULTS: The fast gasotransmitter donors NaHS, sodium nitroprusside and Angeli's salt, administered 5â¯min before the onset of hypoxia, induced an increase in Isc. The response to the subsequently applied hypoxia was characterized by a decrease in Isc, which tended to be reduced only in the presence of the lowest concentration of NaHS (10⯵M) tested. Reoxygenation resulted in a slow increase in Isc, which was unaffected by all donors or inhibitors tested. The stable acetylcholine derivative carbachol (50⯵M) was administered at the end of each hypoxia/reoxygenation cycle to test the secretory capacity of the epithelium. Pretreatment of the tissue with the putative CO donor CORM-2 suppressed the secretory response induced by carbachol. The same was observed when cystathionin-γ-lyase and cystathionin-γ-synthase were inhibited simultaneously. Under both conditions, Gt drastically increased suggesting an impaired tissue integrity. CONCLUSIONS: The present results demonstrate that none of the exogenous gasotransmitter releasing drugs significantly ameliorated the changes in epithelial ion transport during the hypoxia/reoxygenation cycle ex vivo. In contrast, the putative CO donor CORM-2 exerted a toxic effect on the epithelium. The endogenous production of H2S, however, seems to have a protective effect on the mucosal integrity and the epithelial transport functions, which - when inhibited - leads to a loss of the secretory ability of the mucosa. This observation together with the trend for improvement observed with a low concentration of the H2S donor NaHS suggests a moderate protective role of low concentrations of H2S under hypoxic conditions.
Assuntos
Gasotransmissores , Sulfeto de Hidrogênio , Liases , Nitritos , Compostos Organometálicos , Sulfetos , Ratos , Animais , Gasotransmissores/farmacologia , Sulfeto de Hidrogênio/farmacologia , Nitroprussiato , Carbacol , Hipóxia , Transporte de ÍonsRESUMO
ATP-sensitive K(+) (KATP) channels couple the metabolic state of a cell to its electrical activity. They consist of a hetero-octameric complex with pore-forming Kir6.x (Kir6.1, Kir6.2) and regulatory sulfonylurea receptor (SUR) subunits. Functional data indicate that KATP channels contribute to epithelial K(+) currents at colonic epithelia. However, their molecular identity and their properties are largely unknown. Therefore, changes in short-circuit current (I sc) induced by the KATP channel opener pinacidil (5 10(-4) mol l(-1)) were measured in Ussing chambers under control conditions and in the presence of different blockers of KATP channels. The channel subunits expressed by the colonic epithelium were identified by immunohistochemistry and by RT-PCR. The K(+) channel opener, when administered at the serosal side, induced an increase in I sc consistent with the induction of transepithelial Cl(-) secretion after activation of basolateral K(+) channels, whereas mucosal administration of pinacidil resulted in a negative I sc. The increase in I sc evoked by serosal pinacidil was inhibited by serosal administration of glibenclamide (5 10(-4) mol l(-1)) and gliclazide (10(-6) mol l(-1)), but was resistant even against a high concentration (10(-2) mol l(-1)) of tolbutamide. In contrast, none of these inhibitors (administered at the mucosal side) reduced significantly the negative I sc induced by mucosal pinacidil. Instead, pinacidil inhibited Cl(-) currents across apical Cl(-) channels in basolaterally depolarized epithelia indicating that the negative I sc induced by mucosal pinacidil is due to a transient inhibition of Cl(-) secretion. In mRNA prepared from isolated colonic crypts, messenger RNA for both pore-forming subunits, Kir6.1 and Kir6.2, and two regulatory subunits (SUR1 and SUR2B) was found. Expression within the colonic epithelium was confirmed for these subunits by immunohistochemistry. In consequence, KATP channels are present in the basolateral membrane of the colonic epithelium; their exact subunit composition, however, has still to be revealed.
Assuntos
Colo/metabolismo , Mucosa Intestinal/metabolismo , Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Sulfonilureias/metabolismo , Animais , Cloretos/metabolismo , Colo/citologia , Colo/fisiologia , Mucosa Intestinal/fisiologia , Transporte de Íons , Canais KATP/genética , Pinacidil/farmacologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ratos , Ratos Wistar , Receptores de Sulfonilureias/agonistas , Receptores de Sulfonilureias/antagonistas & inibidores , Receptores de Sulfonilureias/genéticaRESUMO
In nonexcitable cells, store-operated Ca(2+) entry is the most important pathway for influx of extracellular Ca(2+) serving as a second messenger in the cytoplasm. The present study investigated the expression, localization and polar distribution of two key components of store-operated Ca(2+) entry identified, e.g., in lymphocytes or epithelial cell lines-STIM1 (stromal interacting molecule 1), working as a Ca(2+) sensor in the endoplasmic reticulum, and Orai1, working as the (or part of the) store-operated Ca(2+) channel in the plasma membrane-in a native intestinal epithelium, i.e., rat colon. Immunohistochemical investigations revealed expression of STIM1 and Orai1 in the rat colonic epithelium. Ca(2+) store depletion led to a translocation of STIM1 both to the basolateral as well as to the apical cell pole as observed by confocal microscopy. A Ca(2+) depletion/repletion protocol was used in Ussing chamber experiments to investigate the contribution of basolateral and apical store-operated Ca(2+) entry to the induction of anion secretion. These experiments revealed that Ca(2+)-dependent anion secretion was induced not only by basolateral Ca(2+) repletion but also, to a lesser extent, by apical Ca(2+) repletion. Both responses were suppressed by La(3+). The effect of basolateral Ca(2+) repletion was significantly inhibited by brefeldin A, a blocker of vesicular transport from the endoplasmic reticulum to the Golgi apparatus. In a final series of experiments, fura-2-loaded HT29/B6 cells were used. A carbachol-induced increase in the cytosolic Ca(2+) concentration was significantly reduced when cells were pretreated with siRNA against STIM1. In conclusion, these results demonstrate that STIM1 as a key component of intracellular Ca(2+) signaling is expressed by rat colonic epithelium and is involved in the regulation not only of basolateral but also of apical Ca(2+) influx.
Assuntos
Cálcio/metabolismo , Colo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Colo/citologia , Citoesqueleto/metabolismo , Feminino , Células HT29 , Humanos , Imuno-Histoquímica , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , RNA Interferente Pequeno , Ratos , Ratos Wistar , Molécula 1 de Interação EstromalRESUMO
OBJECTIVES: The main function of myenteric neurons is the control of gut motility. As we recently showed that nitroxyl (HNO) induces intestinal smooth muscle relaxation, it was of interest to evaluate the effects of this signalling molecule on myenteric neurons in order to distinguish its properties in regard to myocytes. METHODS: Myenteric neurons isolated from the ileum of 4-10 days old rats were used. HNO-induced changes in intracellular concentration of Ca2+ or membrane potential and ion currents were measured using the Ca2+-sensitive fluorescent dye fura-2 AM or by electrophysiological whole-cell recordings, respectively. Changes in intracellular thiol groups pool were evaluated using thiol tracker violet. Angeli's salt was used as HNO donor. RESULTS: The HNO donor Angeli's salt induced a significant increase in the cytosolic Ca2+ concentration at the concentration 50 µM and a membrane hyperpolarization from a resting membrane potential of -56.1 ± 8.0 mV to -63.1 ± 8.7 mV (n=7). Although potassium channels primarily drive membrane potential changes in these cells, outwardly rectifying potassium currents were not significantly affected by 50 µM Angeli's salt. Fast inward sodium currents were slightly but not significantly reduced by HNO. In more sensitive cells, HNO tended to reduce the pool of thiol groups. CONCLUSIONS: As in the case of smooth muscle cells, HNO causes hyperpolarization of myenteric neurons, an effect also associated with an increase in intracellular Ca2+ concentration. Pathways other than activation of potassium currents appear to drive the hyperpolarization evoked by HNO.
Assuntos
Gases , Nitritos , Ratos , Animais , Neurônios , Compostos de Sulfidrila , Motilidade Gastrointestinal , PotássioRESUMO
OBJECTIVES: Reactive oxygen and nitrogen species may be produced during inflammation leading to the formation of NO, H2S or HNO. Enzymes such as iNOS, CSE and CBS might also be responsible for polysulfide production. Since these signalling molecules might have an impact on colonic motility, the aim of this study was to compare their effect on rat colonic slow phasic contractions (SPC). METHODS: Organ bath measurements with strips obtained from rat proximal colon were performed using the polysulfide Na2S3, sodium nitroprusside (NaNP), sodium hydrogen sulfide (NaHS), Angeli's salt as NO, H2S, and HNO donors, respectively. TTX (1 µM) was used to block neuronal activity. RESULTS: All four molecules, concentration-dependently, inhibited the amplitude and frequency of SPC both in the circular and longitudinal muscle layer. The relative potency was NaNP>Angeli's salt>NaHS>Na2S3. The inhibitory response induced by NaNP (1 µM) and Angeli's salt (50 µM) was reversed by ODQ (10 µM) whereas the inhibitory effect of NaHS (1 mM) was reversed by apamin (1 µM) and glibenclamide (10 µM). Na2S3 (1 mM) response was partially reversed by apamin (1 µM) and glibenclamide (10 µM). High concentrations of Na2S3 caused an increase in tone. Low concentrations of NaHS or Na2S3 did not potentiate NaNP responses. CONCLUSIONS: All signalling molecules inhibit SPC in both muscle layers. The effect is independent of neural activity and involves guanylyl cyclase (NO and HNO) and SKCa and KATP channels (NaHS or Na2S3). Other pathways might also be involved in Na2S3 responses. Accordingly, complementary mechanisms of inhibition might be attributable to these signalling molecules.
RESUMO
Gold nanoparticles have a high potential to be a treatment of diseases by their specific drug delivery properties and multivalent receptor stimulation. For the present project, spherical gold nanoparticles were synthesized and functionalized with the muscarinic receptor antagonist atropine (Au-MUDA-AT NPs). The diameter of the gold core could precisely be controlled by using different synthetic methods and reducing agents resulting in functionalized gold nanoparticles with diameters ranging from 8 to 16 nm. The ability to interact with intestinal muscarinic receptors is size-dependent. When using intestinal chloride secretion induced by the stable acetylcholine derivative, carbachol, as read-out, the strongest inhibition, i.e. the most efficient blockade of muscarinic receptors, was observed with 13 nm sized Au-MUDA-AT NPs. Functional experiments indicate that Au-MUDA-AT NPs with a diameter of 14 nm are able to pass the intestinal mucosa in a time-dependent manner after administration to the intestinal lumen. For example, luminally administered Au-MUDA-AT NPs inhibited contractions of the small intestinal longitudinal muscle layer induced by electrical stimulation of myenteric neurons. A similar inhibition of basolateral epithelial receptors was observed after luminal administration of Au-MUDA-AT NPs when using carbachol-induced chloride secretion across the intestinal epithelium as a test system. Thus, Au-MUDA-AT NPs might be a therapeutic tool for the modulation of intestinal secretion and motility after oral application in the future.
RESUMO
In this study, we present a strategy for the synthesis of catecholamine functionalised gold nanoparticles and investigated their multivalent interactions with adrenergic receptors in different biological systems. The catecholamines adrenaline and noradrenaline represent key examples of adrenergic agonists. We used gold nanoparticles as carriers and functionalised them on their surface with a variety of these neurotransmitter molecules. For this purpose, we synthesised each ligand separately using mercaptoundecanoic acid as a bifunctional linking unit and adrenaline (or noradrenaline) as a biogenic amine. This ligand was then immobilised onto the surface of presynthesised spherical monodispersive gold nanoparticles in a ligand exchange reaction. After detailed analytical characterisations, the functionalised gold nanoparticles were investigated for their interactions with adrenergic receptors in intestinal, cardiac and respiratory tissues. Whereas the contractility of respiratory smooth muscle cells (regulated by ß2-receptors) was not influenced, (nor)adrenaline functionalised nanoparticles administered in nanomolar concentrations induced epithelial K+ secretion (mediated via different ß-receptors) and increased contractility of isolated rat cardiomyocytes (mediated by ß1-receptors). The present results suggest differences in the accessibility of adrenergic agonists bound to gold nanoparticles to the binding pockets of different ß-receptor subtypes.
RESUMO
Gaseous molecules such as nitric oxide (NO), hydrogen sulfide (H2S), or carbon monoxide (CO) are involved in the regulation of colonic water and salt transport, which can be switched between absorption and secretion. Nitric oxide is produced from the amino acid L-arginine by different isoforms of the enzyme NO synthase, which are expressed both by enteric neurones and by the colonic epithelium. NO donors evoke a transepithelial Clâ» secretion in vitro. Most actions of NO are mediated by a stimulation of guanosine 5' cyclic monophosphate (cGMP) synthesis via activation of the soluble guanylate cyclase. In rat colon, NO possesses several main action sites: a stimulation of apical Clâ» channels most probably not related to cGMP-dependent phosphorylation, and an increase in the cytosolic Ca²âº concentration, which stimulates a Ca²âº-dependent K⺠conductance in the basolateral membrane. Hydrogen sulfide, produced during the metabolism of the amino acid L-cysteine, also evokes a Clâ» secretion, either by stimulation of secretomotor submucosal neurones as in guinea-pig colon or by activating Ca²âº-dependent and ATP-sensitive K⺠channels as in rat colon. The third gasotransmitter, CO, produced during the degradation of heme, evokes anion secretion carried by Clâ» and HCO3â». This response is mainly caused by the activation of apical anion channels and a stimulation of Ca²âº-dependent K⺠channels via an increase of the cytosolic Ca²âº concentration. Consequently, gaseous molecules produced by enteric neurones, epithelial cells, as well-in the case of H2S-the microbial flora affect key transport enzymes involved in colonic ion transport.
Assuntos
Colo/metabolismo , Bombas de Íon/metabolismo , Transporte de Íons , Animais , Monóxido de Carbono/metabolismo , Colo/enzimologia , Colo/inervação , Humanos , Sulfeto de Hidrogênio/metabolismo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Bombas de Íon/agonistas , Bombas de Íon/antagonistas & inibidores , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
The pharmacological properties of nitroxyl (HNO) donors in the gastrointestinal tract are unknown. We investigated the properties of this molecule in the regulation of gastrointestinal contractility focusing on its possible interaction with other gaseous signaling molecules such as NO and H2S. Organ bath, Ca2+ imaging, and microelectrode recordings were performed on rat intestinal samples, using Angeli's salt as HNO donor. Angeli's salt caused a concentration-dependent relaxation of longitudinal or circular muscle strips of the ileum and the proximal colon. This relaxation was strongly inhibited by the Rho-kinase inhibitor Y-27632 (10 µM), by the reducing agent DTT or by the inhibitor of soluble guanylate cyclase (sGC) ODQ (10 µM) alone or in combination with the inhibitors of the endogenous synthesis of H2S ß-cyano-L-alanine (5 mM) and amino-oxyacetate (5 mM). Preventing endogenous synthesis of NO by the NO synthase inhibitor L-NAME (200 µM) did not affect the relaxation induced by HNO. HNO induced an increase in cytosolic Ca2+ concentration in colonic myocytes. It also elicited myocyte membrane hyperpolarization that amounted to -10.6 ± 1.1 mV. ODQ (10 µM) and Apamin (1 µM), a selective inhibitor of small conductance Ca2+-activated K+ channels (SKca), strongly antagonized this effect. We conclude that HNO relaxes the gastrointestinal tract musculature by hyperpolarizing myocytes via activation of the sGC/cGMP pathway similarly to NO, not only inhibiting the RhoK and activating MLCP as do both NO and H2S but also increasing cytosolic Ca2+ for activation of SK C a contributing to hyperpolarization.
RESUMO
Hydrogen sulfide (H2S) is a well-known environmental chemical threat with an unpleasant smell of rotten eggs. Aside from the established toxic effects of high-dose H2S, research over the past decade revealed that cells endogenously produce small amounts of H2S with physiological functions. H2S has therefore been classified as a "gasotransmitter." A major challenge for cells and tissues is the maintenance of low physiological concentrations of H2S in order to prevent potential toxicity. Epithelia of the respiratory and gastrointestinal tract are especially faced with this problem, since these barriers are predominantly exposed to exogenous H2S from environmental sources or sulfur-metabolising microbiota. In this paper, we review the cellular mechanisms by which epithelial cells maintain physiological, endogenous H2S concentrations. Furthermore, we suggest a concept by which epithelia use their electrolyte and liquid transport machinery as defence mechanisms in order to eliminate exogenous sources for potentially harmful H2S concentrations.
Assuntos
Eletrólitos/metabolismo , Células Epiteliais/metabolismo , Gasotransmissores/metabolismo , Sulfeto de Hidrogênio/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Sulfeto de Hidrogênio/farmacologia , Pulmão/citologiaRESUMO
Ischemia causes severe damage in the gastrointestinal tract. Therefore, it is interesting to study how the barrier and transport functions of intestinal epithelium change under hypoxia and subsequent reoxygenation. For this purpose we simulated hypoxia and reoxygenation on mucosa-submucosa preparations from rat distal colon in Ussing chambers and on isolated crypts. Hypoxia (N2 gassing for 15 min) induced a triphasic change in short-circuit current (Isc): a transient decrease, an increase and finally a long-lasting fall below the initial baseline. During the subsequent reoxygenation phase, Isc slightly rose to values above the initial baseline. Tissue conductance (Gt) showed a biphasic increase during both the hypoxia and the reoxygenation phases. Omission of Cl(-) or preincubation of the tissue with transport inhibitors revealed that the observed changes in Isc represented changes in Cl(-) secretion. The radical scavenger trolox C reduced the Isc response during hypoxia, but failed to prevent the rise of Isc during reoxygenation. All changes in Isc were Ca(2+)-dependent. Fura-2 experiments at loaded isolated colonic crypts revealed a slow increase of the cytosolic Ca(2+) concentration during hypoxia and the reoxygenation phase, mainly caused by an influx of extracellular Ca(2+). Surprisingly, no changes could be detected in the fluorescence of the superoxide anion-sensitive dye mitosox or the thiol-sensitive dye thiol tracker, suggesting a relative high capacity of the colonic epithelium (with its low O2 partial pressure even under physiological conditions) to deal with enhanced radical production during hypoxia/reoxygenation.
RESUMO
The response characteristics of acetylcholinesterase-modified AlGaN/GaN solution-gate field-effect transistors (AcFETs) are quantitatively analyzed by means of a kinetic model. The characterization shows that the covalent enzyme immobilization process yields reproducible AcFET characteristics with a Michaelis constant KM of (122 ± 4) µM for the immobilized enzyme layer. The increase of KM by a factor of 2.4 during the first four measurement cycles is attributed to partial denaturation of the enzyme. The AcFETs were used to record the release of acetylcholine (ACh) by neuronal tissue cultivated on the gate area upon stimulation by rising the extracellular K(+) concentration. The neuronal tissue constituted of isolated myenteric neurons from four to 12 days old Wistar rats, or sections from the muscularis propria containing the myenteric plexus from adult rats. For both cases the AcFET response was demonstrated to be related to the activity of the immobilized acetylcholinesterase using the reversible acetylcholinesterase blocker donepezil. A concentration response curve of this blocking agent revealed a half maximal inhibitory concentration of 40 nM which is comparable to values measured by complementary in vitro methods.
Assuntos
Acetilcolinesterase/metabolismo , Compostos de Alumínio/química , Condutometria/instrumentação , Gálio/química , Plexo Mientérico/metabolismo , Neurônios/enzimologia , Transistores Eletrônicos , Acetilcolinesterase/química , Animais , Técnicas Biossensoriais , Células Cultivadas , Desenho Assistido por Computador , Ativação Enzimática , Enzimas Imobilizadas , Desenho de Equipamento , Análise de Falha de Equipamento , Monitorização Fisiológica/instrumentação , Plexo Mientérico/citologia , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In the emerging era of post-genomic research on schistosomes, new methods are required to functionally analyse genes of interest in more detail. Among other tools, schistosome cell lines are needed to overcome present research constraints. Based on a recently established organ isolation protocol for adult Schistosoma mansoni, we report here on the successful enrichment of vitellarium tissue and isolation of vitelline cells. Morphological analyses performed by bright field, fluorescence, scanning and transmission electron microscopy showed typical features of S1 to S4 stage vitelline cells. In addition, molecular analyses using reverse transcription-PCR confirmed the identity of vitelline cells. Cytological and physiological studies included staining experiments with viability dyes and a neutral lipid stain, as well as calcium (Ca2+) imaging. Together they demonstrated cell viability, the possibility to define the differentiation stage of individual vitelline cells, and the suitability to investigate Ca(2+)-associated processes herein. Finally, fluorescence-activated cell sorting was shown to be a convenient way to separate and enrich S1 to S4 stage vitelline cells. In summary, these results demonstrate the expedience of the organ isolation protocol to obtain vitellarium tissue. Importantly, the protocol allows vitelline cells representing defined differentiation stages to be purified, which can be cultured in vitro and used to investigate diverse aspects of schistosome reproductive biology in the post-genomic era.
Assuntos
Ovário/citologia , Schistosoma mansoni/citologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Técnicas de Cultura de Células , Células Cultivadas , Feminino , Metabolismo dos Lipídeos , Microscopia Eletrônica de TransmissãoRESUMO
Bradykinin is a mediator involved in inflammatory processes in the gut. Here we investigated the effect of bradykinin on the electrical activity of rat myenteric neurons, the key players for regulation of gastrointestinal motility. Bradykinin (2 × 10(-8)mol/l) induced a biphasic increase in frequency of action potentials measured with microelectrode arrays. This increase was mirrored by a biphasic increase in cytosolic Ca(2+) concentration ([Ca(2+)]i), which was observed in about 40% of the myenteric neurons. The bradykinin B1 receptor agonist des-arg(9)-bradykinin as well as the bradykinin B2 receptor agonist hyp(3)-bradykinin induced a similar effect on [Ca(2+)]i. Immunocytochemical stainings confirmed the expression of both receptor types by myenteric ganglionic cells. Real time PCR showed that the inducible B1 receptor was upregulated during cell culture. The inhibition of cyclooxygenases with piroxicam reduced the effect of bradykinin on the electrical activity of myenteric neurons. The suppression of the glial growth on microelectrode arrays did not affect the bradykinin-induced change in frequency of action potentials. This suggests that prostaglandins, which probably mediate the effect of bradykinin, are not exclusively released from glial cells. The bradykinin-induced increase in [Ca(2+)]i was dependent on the presence of extracellular Ca(2+) and was inhibited by Co(2+), Cd(2+), and Ni(2+), blockers of voltage-dependent Ca(2+) channels, indicating a stimulation of the influx of extracellular Ca(2+) by the kinin. Consequently, bradykinin induces a Ca(2+) influx in myenteric neurons via Ca(2+) channels in the plasma membrane.
Assuntos
Bradicinina/farmacologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Plexo Mientérico/citologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Cálcio/metabolismo , Feminino , Masculino , Imagem Molecular , Plexo Mientérico/fisiologia , Neurônios/metabolismo , Prostaglandinas/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor B1 da Bradicinina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The aim of this study was to investigate whether nitroxyl (HNO), a redox variant of the radical gasotransmitter nitric oxide (NO) with therapeutically promising properties, affects colonic ion transport. Changes in short-circuit current (Isc) induced by the HNO donor Angeli's salt were recorded in Ussing chambers. Cytosolic Ca(2+) concentration was measured with fura-2. The nitroxyl donor induced a concentration-dependent increase in Isc across rat distal colon which was due to a stimulation of chloride secretion. The secretion induced by Angeli's salt (5×10(-4)mol/l) was not altered by the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO), but was abolished by the HNO scavenger l-cysteine. The response was not dependent on the activity of soluble guanylate cyclase or enteric neurons, but was inhibited by indomethacin. Experiments with apically permeabilized epithelia revealed the activation of basolateral K(+) channels and a stimulation of the current carried by the basolateral Na(+)-K(+)-pump by Angeli's salt. The secretion induced by Angeli's salt was reduced in the absence of extracellular Ca(2+). A prominent increase in the cytosolic Ca(2+) concentration was evoked by Angeli's salt predominantly in subepithelial cells within the submucosa, which had the same dependence on extracellular Ca(2+) as the Angeli's salt-induced Cl(-) secretion. Consequently, Angeli's salt induces a soluble guanylate cyclase-independent, Ca(2+)-dependent Cl(-) secretion via activation of the Na(+)-K(+)-ATPase and of basolateral K(+) channels. Cyclooxygenase metabolites produced within the submucosa seem to be involved in this response.
Assuntos
Colo/efeitos dos fármacos , Colo/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Nitritos/química , Nitritos/farmacologia , Óxidos de Nitrogênio/química , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Cloretos/metabolismo , Colo/citologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Fura-2/metabolismo , Técnicas In Vitro , Mucosa Intestinal/citologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
The aim of this study was to identify the actions of stimulation of endogenous production of H(2)S by cysteine, the substrate for the two H(2)S-producing enzymes, cystathionine-ß-synthase and cystathionine-γ-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc) induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and ß-cyano-L-alanine, i.e., inhibitors of H(2)S-producing enzymes. In contrast, Na cysteinate evoked a biphasic change in Isc, i.e., an initial fall followed by a secondary increase, which was also reduced by these enzyme inhibitors. All responses were dependent on the presence of Cl(-) and inhibited by bumetanide, suggesting that free cysteine induces an inhibition of transcellular Cl(-) secretion, whereas Na cysteinate - after a transient inhibitory phase - activates anion secretion. The assumed reason for this discrepancy is a fall in the cytosolic pH induced by free cysteine, but not by Na cysteinate, as observed in isolated colonic crypts loaded with the pH-sensitive dye, BCECF. Intracellular acidification is known to inhibit epithelial K(+) channels. Indeed, after preinhibition of basolateral K(+) channels with tetrapentylammonium or Ba(2+), the negative Isc induced by free cysteine was reduced significantly. In consequence, stimulation of endogenous H(2)S production by Na cysteinate causes, after a short inhibitory response, a delayed activation of anion secretion, which is missing in the case of free cysteine, probably due to the cytosolic acidification. In contrast, diallyl trisulfide, which is intracellularly converted to H(2)S, only evoked a monophasic increase in Isc without the initial fall observed with Na cysteinate. Consequently, time course and amount of produced H(2)S seem to strongly influence the functional response of the colonic epithelium evoked by this gasotransmitter.
RESUMO
Complex regional pain syndrome (CRPS) is a painful condition affecting one or more extremities of the body, marked by a wide variety of symptoms and signs that are often difficult to manage because the pathophysiology is incompletely understood. Thus, diverse treatments might be ineffective. A recent report revealed the presence of autoantibodies against differentiated autonomic neurons in CRPS patients. However, it remained unclear how the antibodies act in the development of CRPS. We therefore aimed to characterize these antibodies and identify target antigens. Functional properties of affinity-purified immunoglobulin G of control subjects or CRPS patients were assessed using a cardiomyocyte bioassay. Putative corresponding receptors were identified using antagonistic drugs, and synthesized peptide sequences corresponding to segments of these receptors were used to identify the target epitopes. Chinese hamster ovary cells were transfected with putative receptors to ensure observed binding. Further, changes in the intracellular Ca(2+) concentration induced by agonistic immunoglobulin G were measured using the Ca(2+)-sensitive fluorescent dye fura-2 assay. Herein, we demonstrate the presence of autoantibodies in a subset of CRPS patients with agonistic-like properties on the ß(2) adrenergic receptor and/or the muscarinic-2 receptor. We identified these autoantibodies as immunoglobulin G directed against peptide sequences from the second extracellular loop of these receptors. The identification of functionally active autoantibodies in serum samples from CRPS patients supports an autoimmune pathogenesis of CRPS. Thus, our findings contribute to the further understanding of this disease, could help in the diagnosis in future, and encourage new treatment strategies focusing on the immune system.
Assuntos
Autoanticorpos/fisiologia , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/metabolismo , Síndromes da Dor Regional Complexa/imunologia , Síndromes da Dor Regional Complexa/metabolismo , Receptor Muscarínico M2/imunologia , Receptores Adrenérgicos beta 2/imunologia , Adulto , Animais , Animais Recém-Nascidos , Autoanticorpos/biossíntese , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/fisiologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Ratos , Ratos Wistar , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismoRESUMO
Oxidants, produced e.g. during inflammation, alter gastrointestinal functions finally leading to diarrhoea and/or tissue damage. There is only scarce information about the action of oxidants on enteric neurones, which play a central role in the regulation of many gastrointestinal processes. Therefore, the effect of an oxidant, H(2)O(2), on cultured rat myenteric neurones was studied with the whole-cell patch-clamp and imaging (fura-2) techniques. H(2)O(2) (5 mmol/l) induced an increase in the cytosolic Ca(2+) concentration. Both an intracellular release via IP(3) and ryanodine receptors as well as a Gd(3+)-sensitive Ca(2+) influx contributed to this response. Measurement of the membrane potential revealed that the neuronal membrane hyperpolarized by 11.3+/-0.8 mV in the presence of H(2)O(2). Inhibition of Ca(2+)-dependent K(+) channels prevented this hyperpolarization. Voltage-clamp experiments revealed a second action of the oxidant, i.e. a strong inhibition of the fast Na(+) current responsible for the generation of action potentials. This effect seemed to be mediated by the hydroxyl radical (*OH), as Fe(2+) (100 micromol/l), which leads to the generation of this radical from H(2)O(2) via the Fenton reaction, strongly potentiated the action of an ineffective concentration (100 micromol/l) of the oxidant. Protein phosphorylation/dephosphorylation seems to be involved in the mechanism of action of H(2)O(2), as the protein phosphatase inhibitor calyculin A (100 nmol/l) strongly reduced the inhibition of Na(+) current by H(2)O(2). This effect was mimicked by the protein phosphatase 2A specific inhibitor endothall (100 nmol/l), whereas the PP1 blocker tautomycin (3 nmol/l) was less effective. These results suggest that H(2)O(2) reduces the excitability of rat myenteric neurones by a change of basal membrane potential and an inhibition of Na(+) currents.