RESUMO
How fatty acids stimulate enteroendocrine cells to release cholecystokinin (CCK) is largely unknown. Recently, we proposed that the murine enteroendocrine cell line, STC-1, responds to insoluble fatty acid aggregates rather than fatty acid monomers in solution. This hypothesis led to two testable predictions. First, other insoluble particles of similar size but unrelated to fatty acid may be able to stimulate STC-1 cells in a similar fashion to dodecanoic acid and second, fatty acid sensing in STC-1 cells should be fairly insensitive to chemical modifications of the fatty acid as long as these modifications do not greatly alter the ability of the molecule to form insoluble aggregates. We used several analogues of dodecanoic acid and several varieties of latex microsphere (varying in size and surface charge) to see whether the predictions of our model hold. We found that while there was at least one latex microsphere that could induce CCK secretion and calcium mobilisation in STC-1 cells, there was a very poor correlation between the presence of insoluble aggregates and a cellular response. Instead the most important property, determining the potency of fatty acid analogues as stimulants of CCK secretion, was their amphipathicity. Removal of either the polar head or lipophilic tail completely abolished the ability of a given fatty acid analogue to stimulate STC-1 cells. These data suggested that while fatty acids can stimulate cells as aggregates, they may also be acting in monomeric form with the oil:water partitioning coefficient playing a crucial role. We finally resolved this issue with the observation that the sulfate ion greatly altered the response of STC-1 cells to monomeric dodecanoic acid. In the presence of sulfate, STC-1 cells will only respond to dodecanoic acid aggregates whereas when sulfate is replaced with chloride the cells clearly respond to dodecanoic acid monomers which are completely in solution. In summary, we propose that dodecanoic acid can stimulate STC-1 cells via two separate pathways one involving fatty acid monomers in solution and one involving fatty acid aggregates. Which pathway dominates depends on the presence of sulfate in the extracellular medium.
Assuntos
Cálcio/fisiologia , Colecistocinina/metabolismo , Ácidos Láuricos/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Linhagem Celular , Ácidos Graxos não Esterificados/farmacologia , Cinética , Látex , Camundongos , Microscopia Confocal , Microesferas , Nicardipino/farmacologiaRESUMO
The interlobular duct cells of the guinea-pig pancreas secrete HCO(3)(-) across their luminal membrane into a HCO(3)(-)-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO(3)(-) transport cannot be achieved by luminal Cl-/HCO(3)(-) exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO(3)(-) efflux, we have measured the intracellular potential (V(m)) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl- -25 mM HCO(3)(-) solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (approximately 5 mV) due to activation of electrogenic Na+-HCO(3)(-) cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately -50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO(3)(-) concentration to 125 mM caused a hyperpolarization (approximately 10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl- efflux across the luminal membrane is minimized by the depletion of intracellular Cl- and offset by the hyperpolarizing effects of Na+-HCO(3)(-) cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO(3)(-) that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO(3)(-) to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO(3)(-).
Assuntos
Bicarbonatos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Potenciais da Membrana/fisiologia , Ductos Pancreáticos/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Ânions/farmacologia , Transporte Biológico Ativo/fisiologia , Bucladesina/farmacologia , Eletroquímica , Feminino , Cobaias , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Transporte de Íons/fisiologia , Cinética , Técnicas de Cultura de Órgãos , Ductos Pancreáticos/química , Secretina/farmacologiaRESUMO
BACKGROUND AND AIMS: The extracellular calcium sensing receptor (CaR) plays a key role in the calcium homeostatic system and is therefore widely expressed in tissues involved in calcium metabolism. However, the CaR has also been identified in other tissues where its role is less clear. We have investigated the presence of the CaR in the human pancreas. METHODS: Messenger RNA for the CaR was detected by reverse transcription-polymerase chain reaction and the protein was localised by immunostaining. CaR function was assayed in Capan-1 cells by measuring intracellular calcium and [(3)H] thymidine incorporation. RESULTS: The receptor was highly expressed in human pancreatic ducts. It was also expressed in exocrine acinar cells, in islets of Langerhans, and in intrapancreatic nerves and blood vessels. The CaR was expressed in both normal and neoplastic human tissue samples but was detected in only one of five ductal adenocarcinoma cells lines examined. Experiments on the CaR expressing adenocarcinoma cell line Capan-1 showed that the CaR was functional and was linked to mobilisation of intracellular calcium. Stimulation of the CaR reduced Capan-1 cell proliferation. CONCLUSIONS: We propose that the CaR may play multiple functional roles in the human pancreas. In particular, the CaR on the duct luminal membrane may monitor and regulate the Ca(2+) concentration in pancreatic juice by triggering ductal electrolyte and fluid secretion. This could help to prevent precipitation of calcium salts in the duct lumen. The CaR may also help to regulate the proliferation of pancreatic ductal cells.
Assuntos
Adenocarcinoma/química , Proteínas de Neoplasias/análise , Pâncreas/química , Neoplasias Pancreáticas/química , Receptores de Superfície Celular/análise , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , RNA Mensageiro/análise , Receptores de Detecção de Cálcio , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Isolated interlobular ducts from the guinea-pig pancreas secrete a HCO3--rich fluid in response to secretin. To determine the role of Cl- transporters in this process, intracellular Cl- concentration ([Cl-]i) was measured in ducts loaded with the Cl--sensitive fluoroprobe, 6-methoxy-N-ethylquinolinium chloride (MEQ). [Cl-]i decreased when the luminal Cl- concentration was reduced. This effect was stimulated by forskolin, was not dependent on HCO3- and was not inhibited by application of the anion channel/transporter inhibitor H2DIDS to the luminal membrane. It is therefore attributed to a cAMP-stimulated Cl- conductance, probably the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. [Cl-]i also decreased when the basolateral Cl- concentration was reduced. This effect was not stimulated by forskolin, was largely dependent on HCO3- and was inhibited by basolateral H2DIDS. It is therefore mediated mainly by Cl-/HCO3- exchange. With high Cl- and low HCO3- concentrations in the lumen, steady-state [Cl-]i was 25-35 mM in unstimulated cells. Stimulation with forskolin caused [Cl-]i to increase by approximately 4 mM due to activation of the luminal anion exchanger. With low Cl- and high HCO3- concentrations in the lumen to simulate physiological conditions, steady-state [Cl-]i was 10-15 mM in unstimulated cells. Upon stimulation with forskolin, [Cl-]i fell to approximately 7 mM due to increased Cl- efflux via the luminal conductance. We conclude that, during stimulation under physiological conditions, [Cl-]i decreases to very low levels in guinea-pig pancreatic duct cells, largely as a result of the limited capacity of the basolateral transporters for Cl- uptake. The resulting lack of competition from intracellular Cl- may therefore favour HCO3- secretion via anion conductances in the luminal membrane, possibly CFTR.
Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Cloretos/metabolismo , Ductos Pancreáticos/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Transporte Biológico , Feminino , Cobaias , Homeostase , Técnicas In Vitro , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Concentração Osmolar , Ductos Pancreáticos/efeitos dos fármacos , PerfusãoRESUMO
Fatty acids induce cholecystokinin (CCK) secretion both in humans and from murine enteroendocrine cell lines. In both cases, only fatty acids above a critical acyl chain length (C(10)) are capable of inducing a response. Using the enteroendocrine cell line STC-1, the aim of this study was to determine whether this acyl chain length dependency is related to the fact that longer chain fatty acids are relatively insoluble in aqueous solutions and, if so, whether it is insoluble aggregates of fatty acids rather than free fatty acids which evoke CCK secretion. Solutions of fatty acids (chain length C(8)-C(14)), which were judged by filtration and Zeta sizer measurement to contain no fatty acid aggregates, never evoked CCK secretion from STC-1 cells. Filtering fatty acid solutions (of chain length C(10), C(12) and C(14)) through polytetrafluoroethylene (PTFE) filters (0.45 microm pore size) revealed a narrow concentration range for each acid over which the amount of fatty acid removed from the solution increased sharply due to the formation of fatty acid aggregates. Filtration experiments, in which suspensions of C(10), C(12) and C(14) fatty acids were passed through pore sizes of 0.2, 0.45 or 1.2 microm, suggested that STC-1 cells did not respond to fatty acid aggregates of greater than 1.2 microm, while at least 50 % of the CCK response was mediated by aggregates which were smaller than 0.45 microm. Fatty acids induce CCK secretion from STC-1 cells by elevating intracellular Ca(2+) concentration ([Ca(2+)](i)). We therefore measured the effects on [Ca(2+)](i) of filtered C(10), C(12) and C(14) fatty acids. In all cases, [Ca(2+)](i) responses were closely correlated with CCK secretion. Interestingly, while filtrates of fatty acid solutions evoked CCK secretion and elevated [Ca(2+)](i), freshly prepared solutions of fatty acids at the same concentration as the filtrates did not. This suggested that fatty acid aggregates were not in equilibrium with the solvent after filtration. The observation that the ability of C(10), C(12) and C(14) filtrates to elevate [Ca(2+)](i) decayed with time was consistent with this hypothesis. Furthermore, sonication of the filtrates abolished their ability to elevate [Ca(2+)](i). These data further suggest that it is a physical property of the fatty acid solution (the presence of insoluble fatty aggregates) which is responsible for the observed cellular responses. We conclude that Ca(2+) mobilisation and CCK secretion in STC-1 cells is driven by a signal transduction mechanism that senses insoluble fatty acid aggregates, rather than free fatty acids in solution.
Assuntos
Células Enteroendócrinas/fisiologia , Ácidos Graxos/fisiologia , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Cátions Bivalentes/farmacologia , Linhagem Celular , Colecistocinina/metabolismo , Ácido Cólico/farmacologia , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Filtração , Membranas Intracelulares/metabolismo , Ácidos Láuricos/farmacologia , Camundongos , Concentração Osmolar , Tamanho da Partícula , Solubilidade , Fatores de TempoRESUMO
The human parotid gland secretes much of the bicarbonate that enters the mouth. Prompted by studies of animal models, this study sought evidence for the expression of a functional Na(+)-HCO(3)(-) cotransporter (NBC) in human parotid acinar cells. Microfluorometric measurements of intracellular pH in isolated acini showed that the recovery from an acid load was achieved in part by HCO(3)(-) uptake via a Na(+)-dependent, DIDS-sensitive mechanism. By reverse transcriptase-polymerase chain reaction, a full-length NBC1 clone was obtained showing more than 99% homology with the human pancreatic isoform hpNBC1. Expressed in Xenopus oocytes, the electrogenicity of the transporter was detected as an inwardly directed, Na(+)- and HCO(3)(-)-dependent flux of negative charge. Immunohistochemistry using antibodies raised to NBC1 showed strong staining of the basolateral membrane of the acinar cells. Therefore, it was concluded that a functional electrogenic Na(+)-HCO(3)(-) cotransporter is expressed in the human parotid gland, and that it contributes to pH regulation in the acinar cells and could play a significant part in salivary secretion.
Assuntos
Glândula Parótida/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Adulto , Idoso , Amilorida/farmacologia , Cloreto de Amônio/farmacologia , Animais , Membrana Celular/metabolismo , Clonagem Molecular , Citofotometria , Diuréticos/farmacologia , Feminino , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Masculino , Potenciais da Membrana/fisiologia , Microeletrodos , Pessoa de Meia-Idade , Oócitos/metabolismo , Glândula Parótida/citologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sódio/metabolismo , Sódio/farmacologia , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Simportadores de Sódio-Bicarbonato/metabolismo , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Estatística como Assunto , Xenopus laevisRESUMO
1. HCO3- secretion was investigated in interlobular duct segments isolated from guinea-pig pancreas using a semi-quantitative fluorometric method. Secretagogue-induced decreases in intracellular pH, following blockade of basolateral HCO3- uptake with a combination of amiloride and DIDS, were measured using the pH-sensitive fluoroprobe BCECF. Apparent secretory HCO3- fluxes were calculated from the initial rate of intracellular acidification. 2. In the presence of HCO3-, stimulation with secretin (10 nM) or forskolin (5 microM) more than doubled the rate of intracellular acidification. This effect was abolished in the absence of HCO3-. It was also abolished in the presence of HCO3- when DIDS and NPPB were applied to the luminal membrane by microperfusion. We therefore conclude that the increase in acidification rate is a useful index of secretagogue-induced HCO3- secretion across the luminal membrane. 3. Secretin, cholecystokinin (CCK) and bombesin each stimulated HCO3- secretion in a dose-dependent fashion. They evoked comparable maximal responses at about 10 nM and the EC50 values were 0.5 nM for secretin, 0.2 nM for CCK and 30 pM for bombesin. Acetylcholine (ACh) was also effective, with a maximum effect at 10 microM. 4. The stimulatory effect of CCK was blocked completely by the CCK1 receptor antagonist devazepide but not by the CCK2 receptor antagonist L365,260. The CCK analogue JMV-180 (Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-phenylethyl ester), which is an agonist of the high-affinity CCK1 receptor but an antagonist of the low-affinity receptor, also stimulated HCO3- secretion but with a smaller maximal effect than CCK. JMV-180 partially inhibited the response to a high concentration of CCK but not to a lower concentration, suggesting that both high- and low-affinity states of the CCK1 receptor evoke HCO3- secretion. 5. The stimulatory effect of bombesin was blocked completely by the gastrin-releasing peptide (GRP) receptor antagonist D-Phe6-bombesin(6-13)-methyl ester (BME) but not by the neuromedin B (NMB) receptor antagonist D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH2 (BIM-23127). 6. Secretagogue-evoked fluid secretion was also examined using video microscopy to measure the rate of swelling of ducts whose ends had sealed during overnight culture. Secretin, CCK, bombesin and ACh all evoked fluid secretion with maximal rates of approximately 0.6 nl x min(-1) x mm(-2), and with concentration dependences similar to those obtained for HCO3- secretion. 7. We conclude that CCK, bombesin and ACh stimulate the secretion of a HCO3--rich fluid by direct actions on the interlobular ducts of the guinea-pig pancreas and that these responses are mediated by CCK1 receptors, GRP receptors and muscarinic cholinoceptors, respectively.
Assuntos
Acetilcolina/farmacologia , Bicarbonatos/metabolismo , Bombesina/farmacologia , Colecistocinina/farmacologia , Ductos Pancreáticos/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Algoritmos , Amilorida/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Diuréticos/farmacologia , Cobaias , Técnicas In Vitro , Masculino , Microscopia de Vídeo , Ductos Pancreáticos/efeitos dos fármacos , Perfusão , Secretina/farmacologia , Estimulação QuímicaRESUMO
Aquaporin (AQP) water channels are expressed in a variety of fluid-transporting epithelia and are likely to play a significant role in salivary secretion. Our aim was to identify and localize the aquaporins expressed in human salivary glands. Total RNA was extracted from human parotid, submandibular, sublingual, and labial glands and from human brain. Expression of aquaporin mRNA was assessed by RT-PCR using specific primers for human AQP1, AQP3, AQP4, and AQP5. All four aquaporins were detected by RT-PCR in all of the glands, and the sequences were confirmed after further amplification with nested primers. Cleaned PCR products were then used as (32)P-labeled cDNA probes in a semiquantitative Northern blot analysis using glyceraldehyde-3-phosphate dehydrogenase as reference. Only AQP1, AQP3, and AQP5 mRNAs were present at significant levels. AQP localization was determined by immunohistochemistry on paraffin sections using affinity-purified primary antibodies and peroxidase-linked secondary antibodies. Each salivary gland type showed a broadly similar staining pattern: AQP1 was localized to the capillary endothelium and myoepithelial cells; AQP3 was present in the basolateral membranes of both mucous and serous acinar cells; AQP4 was not detected; and AQP5 was expressed in the luminal and canalicular membranes of both types of acinar cell. We conclude that AQP3 and AQP5 together may provide a pathway for transcellular osmotic water flow in the formation of the primary saliva.
Assuntos
Aquaporinas/análise , Proteínas de Membrana , Glândulas Salivares/química , Anticorpos , Aquaporina 1 , Aquaporina 3 , Aquaporina 4 , Aquaporina 5 , Aquaporinas/genética , Aquaporinas/imunologia , Antígenos de Grupos Sanguíneos , Northern Blotting , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The properties of K+ channels in these cells were studied using patch-clamp methods. Two channels, with conductances of 165+/-13 pS (n=6) and 30+/-1 pS (n=3), were identified in single-channel experiments. In cell-attached patches the reversal potentials were -67+/-8 and -74+/-2 mV for the large and small conductance channel, respectively, suggesting that both channels are K+-selective. The large conductance channel was also shown to be K+-selective in inside-out patches. The open probability (P(o)) of this channel was increased at depolarizing potentials and by increasing intracellular Ca2+ concentration ([Ca2+]i). These properties suggest that the large conductance channel is a 'maxi' Ca2+-activated K+ channel (BK(Ca)). The small conductance channel was not observed in inside-out patches. Carbachol (CCh; 10(-5) M) activated the BK(Ca) channel, but not the small conductance channel, in cell-attached patches. CCh also caused a dose-dependent increase in [Ca2+]i measured by fura-2 in microspectrofluorimetric studies, with a half-maximal response at approximately 3x10(-6) M. Neither isoproterenol (10(-5) M) nor substance P (10(-6) M) affected K+-channel activity or [Ca2+]i. In whole-cell experiments, CCh caused an increase in outward current. Charybdotoxin (10(-7) M), a BK(Ca) blocker, inhibited a large component of the CCh-induced current. A large component of the charybdotoxin-insensitive current may be carried by Ca2+-activated Cl- channels, which were also observed in human parotid acinar cells. The results indicate that BK(Ca) channels make a significant contribution to the whole-cell conductance in human parotid acinar cells.
Assuntos
Agonistas dos Canais de Cloreto , Canais de Cloreto/metabolismo , Glândula Parótida/metabolismo , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Adulto , Idoso , Cálcio/fisiologia , Carbacol/farmacologia , Canais de Cloreto/análise , Agonistas Colinérgicos/farmacologia , Feminino , Humanos , Modelos Lineares , Masculino , Potenciais da Membrana , Pessoa de Meia-Idade , Glândula Parótida/citologia , Técnicas de Patch-Clamp , Canais de Potássio/análise , Espectrometria de FluorescênciaRESUMO
Both the acinar and ductal cells of the pancreas secrete a near-isotonic fluid and may thus be sites of aquaporin (AQP) water channel expression. Northern blot analysis of mRNA from whole rat pancreas revealed high levels of AQP1 and AQP8 expression, whereas lower levels of AQP4 and AQP5 expression were just detectable by RT-PCR Southern blot analysis. Immunohistochemistry showed that AQP1 is localized in the microvasculature, whereas AQP8 is confined to the apical pole of the acinar cells. No labeling of acinar, ductal, or vascular tissue was detected with antibodies to AQP2-7. With immunoelectron microscopy, AQP8 labeling was observed not only at the apical membrane of the acinar cells but also among small intracellular vesicles in the subapical cytoplasm, suggesting that there may be regulated trafficking of AQP8 to the apical plasma membrane. To evaluate the contribution of AQPs to the membrane water permeability, video microscopy was used to measure the swelling of acinar cells in response to hypotonic stress. Osmotic water permeability was reduced by 90% following exposure to Hg(2+). Since AQP8 is confined to the apical membrane, the marked effect of Hg(2+) suggests that other water channels may be expressed in the basolateral membrane.
Assuntos
Aquaporinas/metabolismo , Pâncreas/metabolismo , Algoritmos , Animais , Aquaporinas/efeitos dos fármacos , Northern Blotting , Southern Blotting , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Soluções Hipotônicas , Imuno-Histoquímica , Masculino , Compostos de Mercúrio/farmacologia , Microscopia Imunoeletrônica , Pâncreas/citologia , Pâncreas/ultraestrutura , RNA/biossíntese , RNA/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
1. Fatty acid-induced cholecystokinin (CCK) secretion in humans and from the enteroendocrine cell line STC-1 depends critically on acyl chain length. 2. Therefore we have characterized the relationship between acyl chain length and the potency of the fatty acid to induce CCK secretion and changes in intracellular Ca2+ concentration ([Ca2+]i) in two enteroendocrine cell lines (STC-1 and GLUTag). We found that the potency of the fatty acid was directly proportional to its chain length and therefore inversely proportional to its solubility. 3. In both cell types, the fatty acid-induced rise in [Ca2+]i in response to decanoic acid (C10), dodecanoic acid (C12) and tetradecanoic acid (C14) was significantly reduced in Ca2+-free medium and largely blocked by nicardipine. Intracellular stores also contributed to the overall shape of the [Ca2+]i peak. Thus all the fatty acids tested caused the release of Ca2+ from stores and influx of extracellular Ca2+, presumably through L-type calcium channels. 4. To probe the site of fatty acid action, we studied the distribution of 14C-labelled dodecanoic acid. This label was rapidly and irreversibly accumulated by both cell types, where it became concentrated about 20-fold. Confocal microscopy of a fluorescent analogue of dodecanoic acid clearly demonstrated that it entered the cytosol and was not merely partitioning in the cell membrane. These data indicate that an intracellular action for fatty acid-induced CCK secretion cannot be eliminated. 5. Dodecanoic acid itself, and not a metabolite, is the agent responsible for triggering Ca2+ entry since a non-metabolizable form of dodecanoic acid (2-bromododecanoic acid) was also capable of inducing a rise in [Ca2+]i in both cell types. 6. In conclusion, the rise in [Ca2+]i in STC-1 and GLUTag cells evoked by medium- to long-chain fatty acids results from the triggering of a specific signalling pathway. Whether triggering occurs through activation of a membrane-bound receptor or at an intracellular site remains to be clarified.
Assuntos
Cálcio/metabolismo , Colecistocinina/metabolismo , Células Enteroendócrinas/metabolismo , Ácidos Graxos/metabolismo , Líquido Intracelular/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Ácidos Decanoicos/metabolismo , Ácidos Decanoicos/farmacologia , Relação Dose-Resposta a Droga , Células Enteroendócrinas/citologia , Células Enteroendócrinas/efeitos dos fármacos , Ácidos Graxos/farmacologia , Humanos , Ácidos Láuricos/metabolismo , Ácidos Láuricos/farmacologia , Estrutura Molecular , Ácido Mirístico/metabolismo , Ácido Mirístico/farmacologia , Nicardipino/farmacologia , Soroalbumina Bovina/farmacologiaRESUMO
Permeabilities of the luminal and basolateral membranes of pancreatic duct cells to CO2 and HCO3- were examined in interlobular duct segments isolated from guinea-pig pancreas. Intracellular pH (pHi) was measured by microfluorometry in unstimulated, microperfused ducts loaded with the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). When HCO3-/CO2 was admitted to the bath, pHi decreased transiently as a result of CO2 diffusion and then increased to a higher value as a result of HCO3- uptake across the basolateral membrane by Na+-HCO3- cotransport. When HCO3-/CO2 was admitted to the lumen, pHi again decreased but no subsequent increase was observed, indicating that the luminal membrane was permeable to CO2 but did not allow HCO3- entry to the cells from the lumen. Only when the luminal HCO3- concentration was raised above 125 mM was HCO3- entry detected. The same was true of duct cells stimulated with forskolin. Recovery of pHi from an acid load, induced by exposure to an NH4+ pulse, was dependent on basolateral but not luminal Na+ and could be blocked by basolateral application of methylisobutylamiloride and H2DIDS. This indicates that the Na+-H+ exchangers and Na+-HCO3- cotransporters are located exclusively at the basolateral membrane. In the presence of HCO3-/CO2, substitution of basolateral Cl- with glucuronate caused larger increases in pHi than substitution of luminal Cl-. This suggests that the anion exchanger activity in the basolateral membrane is greater than that in the luminal membrane. We conclude that the luminal and basolateral membranes are both freely permeable to CO2, but while the basolateral membrane has both uptake and efflux pathways for HCO3-, the luminal membrane presents a significant barrier to the re-entry of secreted HCO3-, largely through the inhibition of the luminal anion exchanger by high luminal HCO3- concentrations.
Assuntos
Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Ductos Pancreáticos/metabolismo , Animais , Bicarbonatos/farmacologia , Transporte Biológico Ativo , Dióxido de Carbono/farmacologia , Cloretos/metabolismo , Feminino , Cobaias , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Troca Iônica , Membranas/metabolismo , Ductos Pancreáticos/efeitos dos fármacos , Perfusão , Permeabilidade , Sódio/metabolismoRESUMO
The protective effect of 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid (rebamipide) on gastric mucosa is well established. Here we demonstrate that rebamipide acts on pancreatic acinar cells to generate oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the activation of cholecystokinin subtype 1 (CCK(1)) receptors. At concentrations higher than 5 microM, rebamipide induced [Ca(2+)](i) oscillations in individual fura-2-loaded pancreatic acinar cells. The frequency of oscillations increased with increasing concentrations of rebamipide, while the latency between stimulation of cells and initiation of [Ca(2+)](i) oscillations decreased with increasing concentration. The [Ca(2+)](i) oscillations evoked by rebamipide were inhibited by the CCK(1) receptor antagonist L-364,718 but not by atropine or the CCK(2) receptor antagonist L-365,260 indicating that rebamipide is a nonpeptide CCK(1) receptor agonist.
Assuntos
Alanina/análogos & derivados , Antiulcerosos/farmacologia , Cálcio/metabolismo , Pâncreas/metabolismo , Quinolonas/farmacologia , Receptores da Colecistocinina/metabolismo , Alanina/antagonistas & inibidores , Alanina/farmacologia , Animais , Antiulcerosos/antagonistas & inibidores , Atropina/farmacologia , Benzodiazepinonas/farmacologia , Devazepida/farmacologia , Corantes Fluorescentes , Fura-2 , Técnicas In Vitro , Masculino , Antagonistas Muscarínicos/farmacologia , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Quinolonas/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/efeitos dos fármacosRESUMO
1. The location of purinoceptors in the pancreatic duct and their role in regulating ductal secretion have been investigated by applying ATP and UTP to basolateral and luminal surfaces of pancreatic ducts isolated from the guinea-pig pancreas. 2. Changes in intracellular Ca2+ concentration were measured by microfluorometry in microperfused interlobular duct segments. Fluid and HCO3- secretion were estimated by monitoring luminal pH and luminal volume in sealed duct segments microinjected with BCECF-dextran. 3. Both ATP and UTP (1 microM) caused biphasic increases in intracellular Ca2+ concentration in pancreatic duct cells when applied to either the basolateral or luminal membrane. 4. Luminal application of both ATP and UTP evoked fluid and HCO3- secretion. The maximum response to 1 microM ATP or UTP was about 75 % of that evoked by secretin. By contrast, basolateral application of ATP or UTP inhibited spontaneous secretion by 52 % and 73 %, respectively, and secretin-evoked secretion by 41 % and 38 %, respectively. 5. The data suggest that luminal nucleotides may act in an autocrine or paracrine fashion to enhance ductal secretion while basolateral nucleotides, perhaps released from nerve terminals, may have an inhibitory effect. The fact that both apical and basolateral purinoceptors elevate intracellular Ca2+, but that they have opposite effects on secretion, suggests that additional signalling pathways are involved.
Assuntos
Trifosfato de Adenosina/farmacologia , Bicarbonatos/metabolismo , Ductos Pancreáticos/metabolismo , Animais , Cálcio/metabolismo , Feminino , Cobaias , Técnicas In Vitro , Ductos Pancreáticos/efeitos dos fármacos , Perfusão , Receptores Purinérgicos/efeitos dos fármacos , Secretina/metabolismo , Estimulação Química , Uridina Trifosfato/farmacologiaRESUMO
Aquaporin (AQP) water channels are widely expressed in the membranes of fluid-transporting epithelia. Despite the fact that salivary glands are the site of considerable water movement, relatively little is known about the role of aquaporins in human salivary glands. We have examined the expression of AQP1 in human parotid, sublingual and labial salivary glands. Total RNA was extracted from glandular tissue obtained from surgery or biopsy. The presence of AQP1 mRNA was demonstrated in each of the three glands by RT-PCR using primers specifically designed for human AQP1. The PCR product from the labial gland RNA was further amplified with nested primers and the sequence confirmed by automated fluorescent DNA sequencing. The cleaned first PCR product from these glands was then used as a 32P-labelled hybridization probe in a Northern analysis which confirmed the presence of significant amounts of AQP1 transcript in all three glands. AQP1 expression was also demonstrated in cryosections of human labial glands by immunohistochemistry using peroxidase-linked antibodies. Antibody labelling was most prominent in the capillaries but was also evident in the basal regions of the labial gland acini, and may therefore be associated with the serous demilunes which are believed to be a significant site of fluid movement.
Assuntos
Aquaporinas/genética , Lábio/anatomia & histologia , Glândulas Salivares Menores/metabolismo , Adulto , Aquaporina 1 , Antígenos de Grupos Sanguíneos , Northern Blotting , Água Corporal/metabolismo , Capilares/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Glândula Parótida/metabolismo , Reação em Cadeia da Polimerase , RNA/análise , RNA Mensageiro/análise , Glândulas Salivares Menores/irrigação sanguínea , Análise de Sequência de DNA , Membrana Serosa/metabolismo , Glândula Sublingual/metabolismo , Transcrição GênicaRESUMO
The balance between the concentrations of free ionized Ca2+ and bicarbonate in pancreatic juice is of critical importance in preventing the formation of calcium carbonate stones. How the pancreas regulates the ionic composition and the level of Ca2+ saturation in an alkaline environment such as the pancreatic juice is not known. Because of the tight cause-effect relationship between Ca2+ concentration and lithogenicity, and because hypercalcemia is proposed as an etiologic factor for several pancreatic diseases, we have investigated whether pancreatic tissues express a Ca2+-sensing receptor (CaR) similar to that recently identified in parathyroid tissue. Using reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy, we demonstrate the presence of a CaR-like molecule in rat pancreatic acinar cells, pancreatic ducts, and islets of Langerhans. Functional studies, in which intracellular free Ca2+ concentration was measured in isolated acinar cells and interlobular ducts, show that both cell types are responsive to the CaR agonist gadolinium (Gd3+) and to changes in extracellular Ca2+ concentration. We also assessed the effects of CaR stimulation on physiological HCO3- secretion from ducts by making measurements of intracellular pH. Luminal Gd3+ is a potent stimulus for HCO3- secretion, being equally as effective as raising intracellular cAMP with forskolin. These results suggest that the CaR in the exocrine pancreas monitors the Ca2+ concentration in the pancreatic juice, and might therefore be involved in regulating the level of Ca2+ in the lumen, both under basal conditions and during hormonal stimulation. The failure of this mechanism might lead to pancreatic stone formation and even to pancreatitis.
Assuntos
Cálcio/metabolismo , Pâncreas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Sequência de Bases , Primers do DNA , Imunofluorescência , Gadolínio/metabolismo , Imuno-Histoquímica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Detecção de Cálcio , Receptores de Superfície Celular/genéticaRESUMO
The regulation of acinar cell volume and the properties of intracellular water were investigated in perfused rat mandibular salivary glands by proton nuclear magnetic resonance (NMR) spectroscopy. Using an inversion-recovery pulse sequence, and an extracellular relaxation reagent (10 mM Gd-DTPA) to suppress the proton NMR signal from extracellular water, acinar cell volume (intracellular water content) in unstimulated glands was shown to depend upon Cl- uptake by basolateral Na+-K+-2Cl- cotransport. Muscarinic and beta-adrenoceptor stimulation induced shrinkage and swelling respectively. In pulsed-field-gradient NMR experiments, the diffusion coefficient of intracellular water was found to be more than an order of magnitude smaller than that of extracellular water. Using this intrinsic difference in diffusivity between the two compartments, cell volume regulation was investigated in intact, perfused glands in the absence of relaxation reagents. Using both NMR techniques, acinar cells in perfused glands were observed to behave like simple osmometers in response to anisosmotic media, and did not show the volume regulatory responses described in dissociated acinar cells.
Assuntos
Glândula Submandibular/citologia , Glândula Submandibular/metabolismo , Água/metabolismo , Animais , Proteínas de Transporte/metabolismo , Tamanho Celular/fisiologia , Meios de Contraste , Difusão , Espaço Extracelular/metabolismo , Gadolínio DTPA , Espectroscopia de Ressonância Magnética , Masculino , Osmose , Ratos , Ratos Sprague-Dawley , Simportadores de Cloreto de Sódio-Potássio , Glândula Submandibular/químicaRESUMO
1. Pancreatic HCO3- and fluid secretion were studied by monitoring luminal pH (pHL) and luminal volume simultaneously in interlobular duct segments isolated from guinea-pig pancreas. The secretory rate and HCO3- flux were estimated from fluorescence images obtained following microinjection of BCECF-dextran (70 kDa, 20 microM) into the duct lumen. 2. Ducts filled initially with a Cl--rich solution swelled steadily (2.0 nl min-1 mm-2) when HCO3-/CO2 was introduced, and the luminal pH increased to 8.08. When Cl- was replaced by glucuronate, spontaneous fluid secretion was reduced by 75 %, and pHL did not rise above 7.3. 3. Cl--dependent spontaneous secretion was largely blocked by luminal H2DIDS (500 microM). We conclude that, in unstimulated ducts, HCO3- transport across the luminal membrane is probably mediated by Cl--HCO3- exchange. 4. Secretin (10 nM) and forskolin (1 microM) both stimulated HCO3- and fluid secretion. The final value of pHL (8.4) and the increase in secretory rate (1.5 nl min-1 mm-2) after secretin stimulation were unaffected by substitution of Cl-. 5. The Cl--independent component of secretin-evoked secretion was not affected by luminal H2DIDS. This suggests that a Cl--independent mechanism provides the main pathway for luminal HCO3- transport in secretin-stimulated ducts. 6. Ducts filled initially with a HCO3--rich fluid (125 mM HCO3-, 23 mM Cl-) secreted a Cl--rich fluid while unstimulated. This became HCO3--rich when secretin was applied. 7. Addition of H2DIDS and MIA (10 microM) to the bath reduced the secretory rate by 56 and 18 %, respectively. Applied together they completely blocked fluid secretion. We conclude that basolateral HCO3- transport is mediated mainly by Na+-HCO3- cotransport rather than by Na+-H+ exchange.
Assuntos
Ductos Pancreáticos/metabolismo , Suco Pancreático/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bicarbonatos/metabolismo , Cloretos/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Corantes Fluorescentes , Cobaias , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Membranas/metabolismo , Microscopia de Fluorescência , Ouabaína/farmacologia , SoluçõesRESUMO
To investigate the role of Cl- in the regulation of the basolateral transporters of salivary acinar cells, we have measured cell volume and intracellular pH (pHi) in perfused rat mandibular glands using proton NMR spectroscopy and BCECF fluorometry respectively. When perfusate Cl- was replaced by glucuronate, isethionate, methylsulphate, nitrate or thiocyanate, cell volume decreased slowly by about 15% over a 10-min period. Replacement with bromide, which substitutes for Cl- on the Na+-K+-2Cl- cotransporter, caused only a small (4%) reduction in cell volume. Replacement of Cl- by glucuronate, isethionate or methylsulphate evoked a biphasic increase in pHi consisting of a rapid initial increase followed by a slower secondary rise whose time course was similar to that of cell shrinkage. As judged by the effects of HCO3- omission, 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 1 mM amiloride, the initial rise in pHi was due to Cl-/HCO3- exchange while the secondary rise resulted from activation of Na+/H+ exchange. Although replacement of Cl- by nitrate or thiocyanate also caused cell shrinkage, these substituting anions were less effective in activating the exchanger. Therefore, while the upregulation of the exchanger following Cl- replacement may be due in part to cell shrinkage, there is also evidence for the involvement of an anion-sensitive regulatory mechanism. This would be consistent with the hypothesis that both changes in cell volume and in intracellular Cl- concentration contribute to the up-regulation of the exchanger following muscarinic stimulation.
Assuntos
Tamanho Celular , Cloretos/farmacologia , Glândula Submandibular/citologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Amilorida/farmacologia , Animais , Ânions , Antiporters/metabolismo , Brometos/farmacologia , Antiportadores de Cloreto-Bicarbonato , Glucuronatos/farmacologia , Ácido Glucurônico , Concentração de Íons de Hidrogênio , Ácido Isetiônico/farmacologia , Masculino , Nitratos/farmacologia , Ratos , Ratos Sprague-Dawley , Trocadores de Sódio-Hidrogênio/metabolismo , Glândula Submandibular/metabolismo , Sulfatos/farmacologia , Tiocianatos/farmacologiaRESUMO
1. Short segments of interlobular duct were microdissected from guinea-pig pancreas following enzymatic digestion. After overnight culture, intracellular pH (pH1) and Na+ concentration ([Na+]i) were measured by microfluorometry in duct cells loaded with either the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or the sodium-binding benzofuran isophthalate (SBFI). 2. The transporters responsible for maintaining pHi above equilibrium were investigated by using the NH4Cl pulse technique to acid load the cells. In the absence of HCO3-/CO2, the recovery of pH1 was Na+ dependent, abolished by 0.2 mM amiloride and by 10 microM N-methyl-N-isobutylamiloride and was therefore attributed to Na(+)-H+ exchange. 3. In the presence of HCO3-/CO2, amiloride only partially inhibited the recovery from acid loading. The amiloride-insensitive component was abolished by 0.5 mM H2DIDS and unaffected by depletion of intracellular Cl- and was therefore attributed to Na(+)-HCO3- cotransport. 4. Stimulation with 10 nM secretin did not cause a significant change in pH1 despite a significant increase in HCO3- efflux. However, in the presence of secretin, addition of 0.5 mM H2DIDS caused a decline in pH1 that was three times more rapid than that obtained with 0.2 mM amiloride. 5. In secretin-stimulated ducts, Na+ uptake increased when HCO3-/CO2 was added to the bath and this increase was strongly inhibited by 0.5 mM H2DIDS. 6. We conclude that Na(+)-HCO3- cotransport contributes approximately 75% of the HCO3- taken up by guinea-pig pancreatic duct cells during stimulation with secretin. It is proposed that electrical coupling between HCO3- efflux at the luminal membrane and electrogenic Na(+)-HCO3- cotransport at the basolateral membrane explains why secretin causes little change in pH1.