Muscarinic receptor activity induces an afterdepolarization in a subpopulation of hippocampal CA1 interneurons.
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Cholinergic input to the hippocampus may be involved in important behavioral functions and the pathophysiology of neurodegenerative diseases. Muscarinic receptor activity in interneurons of the hippocampus may play a role in these actions. In this study, we investigated the effects of muscarinic receptor activity on the excitability of different subtypes of interneurons in rat hippocampal CA1. Most interneurons displayed an afterhyperpolarizing potential (AHP) after depolarization by injected current or synaptic stimulation. In the presence of a muscarinic agonist, the AHP of a subset of these interneurons was replaced by an afterdepolarization (ADP), often of sufficient magnitude to evoke action potentials in the absence of further stimulation. The ADP was insensitive to cadmium and low extracellular calcium. It was blocked by low extracellular sodium but not by tetrodotoxin or low concentrations of amiloride. Muscarinic ADPs were sometimes observed in isolation but were often accompanied by depolarizing, hyperpolarizing, or biphasic changes in the membrane potential. Interneurons with muscarinic ADPs were found in all strata of CA1 and did not fall into a single morphological classification. The potential functions of the prolonged action potential output of interneurons produced by the ADP could include changes in hippocampal circuit properties and facilitation of the release of peptide cotransmitters in these interneurons. (+info)
Ion transport across the normal and CF neonatal murine intestine.
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Neonatal mice with cystic fibrosis (CF) exhibit a very high mortality due to intestinal obstruction localized primarily to the ileum and colon. It has been hypothesized that lack of Cl(-) secretion and possibly elevated Na(+) absorption contribute to the gut problems in CF neonates. Therefore, intestines (ileum, proximal colon, and distal colon) from normal and CF day-old mouse pups were studied on ultra-small-aperture (0.0135 cm(2)) Ussing chambers. All three regions of the normal neonatal intestine responded to forskolin with an increase in short-circuit current, which was completely absent in the CF intestine. The neonatal distal colon exhibited a high rate of amiloride-sensitive electrogenic Na(+) absorption, which did not differ between the normal and CF preparations. The ileum and proximal colon of both genotypes exhibited a small but significant electrogenic Na(+) absorption. The neonatal proximal colon and ileum also exhibited electrogenic Na(+)-glucose cotransport, which was significantly greater in the normal compared with the CF ileum. In addition, all three intestinal regions exhibited electrogenic Na(+)-alanine cotransport, which was significantly reduced in two of the regions of the CF neonatal intestine. It is speculated that: 1) the reduced rate of Na(+)-nutrient cotransport in the CF intestine contributes to the lower rate of growth in CF pups, whereas 2) the elevated electrogenic Na(+) absorption in the neonatal intestine, coupled with an inability to secrete Cl(-), contributes to the intestinal obstruction in the CF pups. (+info)
Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells.
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The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin-dependent uptake mechanisms, endocytic pathways involving clathrin-coated pits, and the mannose receptor. Furthermore, co-localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established. (+info)
Essential requirement of cytosolic phospholipase A(2) for activation of the H(+) channel in phagocyte-like cells.
(52/2048)
The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA(2) in the regulation of both the H(+) channel and the Na(+)/H(+) antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me(2)SO or 5 x 10(-8) M 1, 25-dihydroxyvitamin D(3). Activation of differentiated PLB cells resulted in a Zn(2+)-sensitive alkalization, indicating H(+) channel activity. In contrast, differentiated PLB-D cells failed to activate the H(+) channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA(2) in H(+) channel activation. The presence of the H(+) channel inhibitor Zn(2+) caused significant inhibition of NADPH oxidase activity, suggesting a role of the H(+) channel in regulating oxidase activity. Na(+)/H(+) antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA(2) does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA for activation of the H(+) channel and suggest the participation of this channel in the regulation of NADPH oxidase activity. (+info)
Block of the helix FMRFamide-gated Na+ channel by FMRFamide and its analogues.
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1. In Helix neurones high doses of Phe-Met-Arg-Phe-NH2 (FMRFamide) often evoke biphasic inward whole-cell currents with brief application, and suppression of the current with prolonged application. With outside-out patches, a transient early suppression of the unitary current amplitude was seen following application of high doses of FMRFamide. 2. Continuous application of a concentration of FMRFamide from 30 microM to 1 mM resulted in a reduction in the amplitude of the unitary currents and an increase in open state noise. There was also an increase in the occurrence of smaller, 'subconductance' currents with the higher concentrations of FMRFamide. Similar effects were seen with FMRFamide on FaNaC expressed in oocytes. The FMRFamide analogues FLRFamide and WnLRFamide were more effective in evoking the lower conductance state. These effects of agonist at high concentrations were voltage dependent suggesting channel block. 3. A similar effect was seen when one of the related peptides FKRFamide, FM(D)RFamide, nLRFamide or N-AcFnLRFamide was co-applied with a low FMRFamide concentration. However, the non-amidated peptides FKRF, FLRF and nLRF and also WMDFamide did not have this effect. 4. The inhibition of unitary currents induced by amiloride was qualitatively different from that produced by FMRFamide analogues with no obvious occurrence of subconductance levels. FMRFamide-gated channels were also blocked by guanidinium, but only at very high concentrations. 5. The results strongly suggest a partial inhibition of current flow through the FMRFamide- gated channel by some part of the agonist or the related antagonist peptide molecules. (+info)
Role of endogenous cortisol in basal liquid clearance from distal air spaces in adult guinea-pigs.
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1. We investigated the role of endogenous cortisol in the modulation of distal air space liquid clearance in adult guinea-pigs. Cortisol synthesis was inhibited with the 11-beta-hydroxylase inhibitor metyrapone (0-7 days pretreatment). After cortisol synthesis inhibition, distal air space liquid clearance was measured by the increase in concentration of an instilled 5 % albumin solution after 1 h. 2. Two days of metyrapone pretreatment resulted in a 46+/-19 % decrease in plasma cortisol levels compared with control, which was paralleled by a 60+/-13 % decrease in distal air space liquid clearance. The Na+ channel inhibitor amiloride inhibited 40+/-22 % of distal air space liquid clearance in control animals but did not inhibit distal air space liquid clearance in the metyrapone-pretreated group. Co-injection of dexamethasone prevented the inhibition by metyrapone and the amiloride sensitivity of distal air space liquid clearance was greater than in control animals. After 7 days of metyrapone pretreatment, plasma cortisol levels and distal air space liquid clearance were not significantly different from normal, but amiloride sensitivity was greater than in control animals (91+/-37%). 3. Pretreatment with emetine, a protein synthesis inhibitor, reduced distal air space liquid clearance in control animals and in dexamethasone-co-injected animals, but failed to inhibit distal air space liquid clearance after metyrapone pretreatment. Expression of the epithelial sodium channel alpha-subunit (alphaENaC) mRNA in lung tissue was decreased after 2 days of metyrapone pretreatment and after 7 days pretreatment or after co-injection with dexamethasone, alphaENaC mRNA expression was restored towards control levels. 4. Thus, endogenous cortisol is important for maintaining normal liquid balance in the adult guinea-pig lung and a critical regulatory pathway is by modulation of ENaC expression and/or function. (+info)
Pharmacological inhibition of the Na(+)/Ca(2+) exchanger enhances depolarizations induced by oxygen/glucose deprivation but not responses to excitatory amino acids in rat striatal neurons.
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BACKGROUND AND PURPOSE: Neuronal Na(+)/Ca(2+) exchanger plays a relevant role in maintaining intracellular Ca(2+) and Na(+) levels under physiological and pathological conditions. However, the role of this exchanger in excitotoxicity and ischemia-induced neuronal injury is still controversial and has never been studied in the same neuronal subtypes. METHODS: We investigated the effects of bepridil and 3',4'-dichlorobenzamil (DCB), 2 blockers of the Na(+)/Ca(2+) exchanger, in rat striatal spiny neurons by utilizing intracellular recordings in brain slice preparations to compare the action of these drugs on the membrane potential changes induced either by oxygen and glucose deprivation (OGD) or by excitatory amino acids (EAAs). RESULTS: Bepridil (3 to 100 micromol/L) and DCB (3 to 100 micromol/L) caused a dose-dependent enhancement of the OGD-induced depolarization measured in striatal neurons. The EC(50) values for these effects were 31 micromol/L and 29 micromol/L, respectively. At these concentrations neither bepridil nor DCB altered the resting membrane properties of the recorded cells (membrane potential, input resistance, and current-voltage relationship). The effects of bepridil and DCB on the OGD-induced membrane depolarization persisted in the presence of D-2-amino-5-phosphonovalerate (50 micromol/L) plus 6-cyano-7-nitroquinoxaline-2,3-dione (20 micromol/L), which suggests that they were not mediated by an enhanced release of EAAs. Neither tetrodotoxin (1 micromol/L) nor nifedipine (10 micromol/L) affect the actions of these 2 blockers of the Na(+)/Ca(2+) exchanger, which indicates that voltage-dependent Na(+) channels and L-type Ca(2+) channels were not involved in the enhancement of the OGD-induced depolarization. Conversely, the OGD-induced membrane depolarization was not altered by 5-(N, N-hexamethylene) amiloride (1 to 3 micromol/L), an inhibitor of the Na(+)/H(+) exchanger, which suggests that this antiporter did not play a prominent role in the OGD-induced membrane depolarization recorded from striatal neurons. Bepridil (3 to 100 micromol/L) and DCB (3 to 100 micromol/L) did not modify the amplitude of the excitatory postsynaptic potentials evoked by cortical stimulation. Moreover, these blockers did not affect membrane depolarizations caused by brief applications of glutamate (0.3 to 1 mmol/L), AMPA (0. 3 to 1 micromol/L), and NMDA (10 to 30 micromol/L). CONCLUSIONS: These results provide pharmacological evidence that the activation of the Na(+)/Ca(2+) exchanger exerts a protective role during the early phase of OGD in striatal neurons, although it does not shape the amplitude and the duration of the electrophysiological responses of these cells to EAA. (+info)
Development of an endogenous epithelial Na(+)/H(+) exchanger (NHE3) in three clones of caco-2 cells.
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Expression of endogenous Na(+)/H(+) exchangers (NHEs) NHE3 and NHE1 at the apical (AP) and basolateral (BL) membrane domains was investigated in three clones (ATCC, PF-11, and TC-7) derived from the human adenocarcinoma cell line Caco-2. In all three clones, NHE1 was the only isoform detected at the BL domain during 3 to 22 postconfluent days (PCD). In clone PF-11, the BL NHE1 activity increased up to 7 PCD and remained stable thereafter. Both NHE1 and NHE3 were found at the AP domain at 3 PCD and contributed 67 and 33% to the total AP Na(+)/H(+) exchange, respectively. The AP NHE3 activity increased significantly from 3 to 22 PCD, from 93 to 450 microM H(+)/s, whereas AP NHE1 activity decreased from 192 to 18 microM H(+)/s during that time. Similar results were obtained with the ATCC clone, whereas very little AP NHE3 activity was observed in clone TC-7. Surface biotinylation and indirect immunofluorescence confirmed these results and also suggested an increase in the number of cells expressing NHE3 being the major mechanism of the observed overall increase in NHE3 activity in PF-11 and ATCC clones. Phorbol 12-myristate 13-acetate (PMA, 1 microM) acutely inhibited NHE3 activity by 28% of control, whereas epidermal growth factor (EGF, 200 ng/ml) stimulated the activity by 18%. The effect of PMA was abolished by the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, suggesting involvement of PKC in the PMA-induced inhibition of NHE3. Similar magnitude of inhibition by PMA and stimulation by EGF was observed at 7 and 17 PCD, suggesting the development of regulatory mechanisms in the early postconfluent period. Taken together, these data suggest a close similarity of membrane targeting and regulation of endogenous NHE3 between Caco-2 cells and native small intestinal epithelial cells and support the usefulness of some Caco-2 cell clones as an in vitro model for studies on physiology of NHE3 in the intestinal epithelium. (+info)