Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl- secretion in airway epithelia

We investigated putative mechanisms by which nitric oxide modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in epithelial cells. Immunoprecipitation followed by Western blotting, as well as immunocytochemical and cell surface biotinylation measurements, showed that incubation of both stably transduced (HeLa) and endogenous CFTR expressing (16HBE14o-, Calu-3, and mouse tracheal epithelial) cells with 100 microm diethylenetriamine NONOate (DETA NONOate) for 24-96 h decreased both intracellular and apical CFTR levels. Calu-3 and mouse tracheal epithelial cells, incubated with DETA NONOate but not with 100 microm 8-bromo-cGMP for 96 h, exhibited reduced cAMP-activated short circuit currents when mounted in Ussing chambers. Exposure of Calu-3 cells to nitric oxide donors resulted in the nitration of a number of proteins including CFTR. Nitration was augmented by proteasome inhibition, suggesting a role for the proteasome in the degradation of nitrated proteins. Our studies demonstrate that levels of nitric oxide that are likely to be encountered in the vicinity of airway cells during inflammation may nitrate CFTR resulting in enhanced degradation and decreased function. Decreased levels and function of normal CFTR may account for some of the cystic fibrosis-like symptoms that occur in chronic inflammatory lung diseases associated with increased NO production.     

Cellular mechanism for potentiation of Ca2+-mediated Cl- secretion by the flavonoid baicalein in intestinal epithelia

Flavonoids belong to a large group of plant polyphenols that are consumed daily in large amounts. Our previous findings have shown that baicalein, a major flavonoid derived from the medicinal herb Scutellariae radix, induces Cl(-) secretion across rat colonic mucosa. The current study examines the effect of baicalein on Cl(-) secretion in human colonic epithelial (T84) cells and its interaction with Ca(2+)- and cAMP-dependent secretagogues. We have employed a technique that allows concurrent monitoring of short-circuit current (I(SC)) and [Ca(2+)](i) in polarized epithelium. Basolateral application of baicalein induced a concentration-dependent increase in I(SC). The increase in I(SC) was because of Cl(-) secretion and was not accompanied by any discernible increase in [Ca(2+)](i). Baicalein acted synergistically with Ca(2+)- but not cAMP-dependent secretagogues. In the presence of baicalein, the carbachol and histamine induced increases in I(SC) that were markedly potentiated while increases in [Ca(2+)](i) were not significantly enhanced. Baicalein treatment uncoupled Cl(-) secretion from inhibitory effects normally generated by muscarinic activation. Baicalein treatment also resulted in increased cAMP content and activated PKA activity. Nystatin permeabilization studies revealed that baicalein stimulated an apical Cl(-) current but did not activate any basolateral K(+) current. These data suggest that baicalein potentiates Ca(2+)-mediated Cl(-) secretion through a signaling pathway involving cAMP and protein kinase A, most likely through the cystic fibrosis transmembrane conductance regulator in the apical membrane.     

ENaC inhibition stimulates Cl- secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption. Since ENaC does not transport Cl(-), the purpose of this study was to determine how ENaC modulates Cl(-) absorption. Thus, Cl(-) absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl(-) absorption or transepithelial voltage (V(T)). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na(+)-K(+)-ATPase inhibitor to the bath reduced Cl(-) absorption by ～66-75% and nearly obliterated lumen-negative V(T). In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1a(loxlox)). Whereas benzamil-sensitive Cl(-) absorption did not depend on CFTR, application of a Na(+)-K(+)-2Cl(-) cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl(-) absorption, although the benzamil-sensitive component of V(T) was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, whereas thiazide-sensitive Cl(-) absorption is undetectable. Second, benzamil-sensitive Cl(-) absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V(T). Finally, benzamil-sensitive Cl(-) flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.     

Evidence for basolateral Cl- channels as modulators of apical Cl- secretion in pulmonary epithelia of Xenopus laevis

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl(-) transport. The present study focuses on the function of basolateral Cl(-) channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents (I(SC)) were monitored. Basolateral application of the Cl(-) channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I(SC), indicating a DPC-sensitive Cl(-) conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl(-) gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl(-) conductance was influenced by procedures interfering with apical Cl(-) secretion. For example, the effect of forskolin was increased when basolateral Cl(-) channels were blocked by the simultaneous application of DPC. Activation of apical Cl(-) secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl(-) secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I(SC). Furthermore, inhibition of basolateral anion exchangers responsible for Cl(-) uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl(-) channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl(-) secretion involving a novel Cl(-) recycling mechanism across the basolateral membrane.     

Flagellin-stimulated Cl- secretion and innate immune responses in airway epithelia: role for p38

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10(-7) M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl(-) secretion (I(Cl)) beginning after 3-10 min, reaching a plateau after 20-45 min at DeltaI(Cl) = 15-50 microA/cm(2). Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated I(Cl) in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1beta (produced by both epithelia and neutrophils during infections) stimulated I(Cl) similar to flagellin. Flagellin-, IL-1beta-, ATP-, and forskolin-stimulated I(Cl) were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1beta altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-kappaB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1beta-stimulated I(Cl) by 33-50% but had smaller effects on IL-8 and NF-kappaB. It is concluded that: 1) flagellin and IL-1beta activated p38, NF-kappaB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating I(Cl) and IL-8 but not NF-kappaB; and 3) p38 was more important in flagellin- than IL-1beta-stimulated responses. During P. aeruginosa infections, flagellin and IL-1beta are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-kappaB, and IL-8 would persist.     

Fenofibrate inhibits intestinal Cl- secretion by blocking basolateral KCNQ1 K+ channels

Fibrates are peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands in widespread clinical use to lower plasma triglyceride levels. We investigated the effect of fenofibrate and clofibrate on ion transport in mouse intestine and in human T84 colonic adenocarcinoma cells through the use of short-circuit current (I(sc)) and ion flux analysis. In mice, oral administration of fenofibrate produced a persistent inhibition of cAMP-stimulated electrogenic Cl(-) secretion by isolated jejunum and colon without affecting electroneutral fluxes of (22)Na(+) or (86)Rb(+) (K(+)) across unstimulated colonic mucosa. When applied acutely to isolated mouse intestinal mucosa, 100 microM fenofibrate inhibited cAMP-stimulated I(sc) within 5 min. In T84 cells, fenofibrate rapidly inhibited approximately 80% the Cl(-) secretory responses to forskolin (cAMP) and to heat stable enterotoxin STa (cGMP) without affecting the response to carbachol (Ca(2+)). Both fenofibrate and clofibrate inhibited cAMP-stimulated I(sc) with an IC(50) approximately 1 muM, whereas other PPARalpha activators (gemfibrozil and Wy-14,643) were without effect. Membrane permeabilization experiments on T84 cells indicated that fenofibrate inhibits basolateral cAMP-stimulated K(+) channels (putatively KCNQ1/KCNE3) without affecting Ca(2+)-stimulated K(+) channel activity, whereas clofibrate inhibits both K(+) pathways. Fenofibrate had no effect on apical cAMP-stimulated Cl(-) channel activity. Patch-clamp analysis of HEK-293T cells confirmed that 100 microM fenofibrate rapidly inhibits K(+) currents associated with ectopic expression of human KCNQ1 with or without the KCNE3 beta-subunit. We conclude that fenofibrate inhibits intestinal cAMP-stimulated Cl(-) secretion through a nongenomic mechanism that involves a selective inhibition of basolateral KCNQ1/KCNE3 channel complexes. Our findings raise the prospect of fenofibrate as a safe and effective antidiarrheal agent.     

Ammonium effects on colonic Cl- secretion: anomalous mole fraction behavior

A significant amount of ammonium (NH4+) is absorbed by the colon. The nature of NH4+ effects on transport and NH4+ transport itself in colonic epithelium is poorly understood. The goal of this study was to elucidate the effects of NH4+ on cAMP-stimulated Cl- secretion in the colonic cell line T84. In HEPES-buffered solutions, application of basolateral NH4+ resulted in a reduced level of Cl- secretory current. The effect of NH4+ appears to occur by at least three mechanisms: 1) basolateral membrane depolarization, 2) a competitive effect with K+, and 3) a long-term (>20 min) increase in transepithelial resistance (TER). The competitive effect with K+ exhibits anomalous mole fraction behavior. Transepithelial current relative to that in 10 mM basolateral K+ was inhibited 15% by 10 mM NH4+ alone and by 30% with a mixture of 2 mM K+ and 8 mM NH4+. A mole fraction mix of 2 mM K+:8 mM NH4+ produced a greater inhibition of basolateral membrane K+ current than pure K+ or NH4+ alone. Similar anomalous behavior was also observed for inhibition of bumetanide-sensitive 36Cl- uptake, e.g., Na+-K+-2Cl- -cotransporter (NKCC-1). No anomalous effect was observed on Na+-K+-ATPase current. Both NKCC-1 and Na+-K+-ATPase activity were elevated in 10 mM NH4+ with respect to 10 mM K+. The effect on TER did not exhibit anomalous mole fraction behavior. The overall effect of basolateral NH4+ on cAMP-stimulated transport is dependent on the [K+]o /[NH4+]o ratio at the basolateral membrane, where o is outside of the cell.     

Vasoactive intestinal peptide-stimulated Cl- secretion: activation of cAMP-dependent K+ channels

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.     

cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1

Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.     

Acid-base effects on intestinal Cl- absorption and vesicular trafficking

In rat ileum and colon, apical membrane exchange and net Cl^- absorption are stimulated by increases in PCO₂ or . Because changes in PCO₂ stimulate colonic Na⁺ absorption, in part, by modulating vesicular trafficking of the Na⁺/H⁺ exchanger type 3 isoform to and from the apical membrane, we examined whether changes in PCO₂ affect net Cl^- absorption by modulating vesicular trafficking of the exchanger anion exchanger (AE)1. Cl^- transport across rat distal ileum and colon was measured in the Ussing chamber, and apical membrane protein biotinylation of these segments and Western blots of recovered proteins were performed. In colonic epithelial apical membranes, AE1 protein content was greater at PCO₂ 70 mmHg than at PCO₂ 21 mmHg but was not affected by pH changes in the absence of CO₂. AE1 was internalized when PCO₂ was reduced and exocytosed when PCO₂ was increased, and both mucosal wortmannin and methazolamide inhibited exocytosis. Wortmannin also inhibited the increase in colonic Cl^- absorption caused by an increase in PCO₂. Increases in PCO₂ stimulated ileal Cl^- absorption, but wortmannin was without effect. Ileal epithelial apical membrane AE1 content was not affected by PCO₂. We conclude that CO₂ modulation of colonic, but not ileal, Cl^- absorption involves effects on vesicular trafficking of AE1. PCO₂; ileum; colon; anion exchanger 1; Na⁺/H⁺ exchanger type 3     

Homeostatic regulation of intestinal villous epithelia by B lymphocytes

The epithelial cell of the small intestine is one of the most rapidly regenerating cells in the body. However, the cellular mechanism and biological significance underlying this rapid regeneration remain elusive. In this study we examined the intestinal epithelia of mutant mice that lack B and/or T cells and those of normal littermates. The absence of B cells in Ig mu-chain mutant mice or B and T cells in recombination-activating gene (RAG)-2(-/-) as well as SCID mutant mice was associated with a marked acceleration of epithelial cell turnover and an up-regulation of the expression of MHC class II molecules. No such effects were observed in T cell-deficient TCR-delta and -beta double-mutant mice. As far as the goblet cells of villous epithelium are concerned, absolute numbers of them remained the same among these mutant mice that have no B and/or T cells. Alymphoplasia (aly/aly) mutant mice that lacked Peyer's patches and Ig-producing cells in the lamina propria, but harbored a large number of intestinal mucosal T cells, also displayed a significant acceleration of epithelial cell turnover and, to some extent, up-regulated expression of MHC class II molecules. Notably, the accelerated epithelial cell turnover was not observed and returned to normalcy in the Ig mu-chain mutant mice that had been given antibiotic-containing water. These findings indicate that B cells down-regulate the generation and differentiation of intestinal epithelial cells in the normal wild-type condition and suggest that enteric microorganisms are implicated in the accelerated generation of epithelial cells in mice that have no B cells.     

Bestrophin-1 enables Ca2+-activated Cl- conductance in epithelia

Epithelial cells express calcium-activated Cl(-) channels of unknown molecular identity. These Cl(-) channels play a central role in diseases such as secretory diarrhea, polycystic kidney disease, and cystic fibrosis. The family of bestrophins has been suggested to form calcium-activated Cl(-) channels. Here, we demonstrate molecular and functional expression of bestrophin-1 (BEST1) in mouse and human airways, colon, and kidney. Endogenous calcium-activated whole cell Cl(-) currents coincide with endogenous expression of the Vmd2 gene product BEST1 in murine and human epithelial cells, whereas calcium-activated Cl(-) currents are absent in epithelial tissues lacking BEST1 expression. Blocking expression of BEST1 with short interfering RNA or applying an anti-BEST1 antibody to a patch pipette suppressed ATP-induced whole cell Cl(-) currents. Calcium-dependent Cl(-) currents were activated by ATP in HEK293 cells expressing BEST1. Thus, BEST1 may form the Ca2+-activated Cl(-) current, or it may be a component of a Cl(-) channel complex in epithelial tissues.     

Structural basis for physiological regulation of paracellular pathways in intestinal epithelia

Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10-25 mM glucose, alanine or leucine. Resistances of intercellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01-10 kHz. The segments were then fixed in situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixed in situ at 38 degrees C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.     

Structural basis for physiological regulation of paracellular pathways in intestinal epithelia

Summary Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10–25mm glucose, alanine or leucine. Resistances of inter-cellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01–10 kHz. The segments were then fixedin situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixedin situ at 38°C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.     

A novel role for defensins in intestinal homeostasis: regulation of IL-1beta secretion

Impaired expression of alpha-defensin antimicrobial peptides and overproduction of the proinflammatory cytokine IL-1beta have been associated with inflammatory bowel disease. In this study, we examine the interactions between alpha-defensins and IL-1beta and the role of defensin deficiency in the pathogenesis of inflammatory bowel disease. It was found that matrix metalloproteinase-7-deficient (MMP-7(-/-)) mice, which produce procryptdins but not mature cryptdins (alpha-defensins) in the intestine, were more susceptible to dextran sulfate sodium-induced colitis. Furthermore, both baseline and dextran sulfate sodium-induced IL-1beta production in the intestine were significantly up-regulated in MMP-7(-/-) mice compared with that in control C57BL/6 mice. To elucidate the molecular mechanism for the increased IL-1beta production in defensin deficiency in vivo, we evaluated the effect of defensins on IL-1beta posttranslational processing and release. It was found that alpha-defensins, including mouse Paneth cell defensins cryptdin-3 and cryptdin-4, human neutrophil defensin HNP-1, and human Paneth cell defensin HD-5, blocked the release of IL-1beta from LPS-activated monocytes, whereas TNF-alpha expression and release were not affected. Unlike alpha-defensins, human beta-defensins and mouse procryptdins do not have any effect on IL-1beta processing and release. Thus, alpha-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1beta.     

Uncoupling protein-1: involvement in a novel pathway for beta-adrenergic,cAMP-mediated intestinal relaxation

The pathway for adrenergic relaxation of smooth muscle is not fully understood. As mitochondrial uncoupling protein-1 (UCP1) expression has been reported in cells within the longitudinal smooth muscle layer of organs exhibiting peristalsis, we examined whether the absence of UCP1 affects adrenergic responsiveness. Intestinal (ileal) segments were obtained from UCP1-ablated mice and from wild-type mice (C57Bl/6, 129/SvPas, and outbred NMRI). In UCP1-containing mice, isoprenaline totally inhibited contractions induced by electrical field stimulation, but in intestine from UCP1-ablated mice, a significant residual contraction remained even at a high isoprenaline concentration; the segments were threefold less sensitive to isoprenaline. Also, when contraction was induced by carbachol, there was a residual isoprenaline-insensitive contraction. Similar results were obtained with the beta(3)-selective agonist CL-316,243 and with the adenylyl cyclase stimulator forskolin. Thus the UCP1 reported to be expressed in the longitudinal muscle layer of the mouse intestine is apparently functional, and UCP1, presumably through uncoupling, may be involved in a novel pathway leading from increased cAMP levels to relaxation in organs exhibiting peristalsis.