Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency

Malfunctions of potassium channels are increasingly implicated as causes of neurological disorders. However, the functional roles of the large-conductance voltage- and Ca(2+)-activated K(+) channel (BK channel), a unique calcium, and voltage-activated potassium channel type have remained elusive. Here we report that mice lacking BK channels (BK(-/-)) show cerebellar dysfunction in the form of abnormal conditioned eye-blink reflex, abnormal locomotion and pronounced deficiency in motor coordination, which are likely consequences of cerebellar learning deficiency. At the cellular level, the BK(-/-) mice showed a dramatic reduction in spontaneous activity of the BK(-/-) cerebellar Purkinje neurons, which generate the sole output of the cerebellar cortex and, in addition, enhanced short-term depression at the only output synapses of the cerebellar cortex, in the deep cerebellar nuclei. The impairing cellular effects caused by the lack of postsynaptic BK channels were found to be due to depolarization-induced inactivation of the action potential mechanism. These results identify previously unknown roles of potassium channels in mammalian cerebellar function and motor control. In addition, they provide a previously undescribed animal model of cerebellar ataxia.     

Transporter-mediated bile acid uptake causes Ca2+-dependent cell death in rat pancreatic acinar cells

BACKGROUND & AIMS: The mechanism by which cholelithiasis increases the risk of acute pancreatitis remains obscure. Because bile acids can enter the pancreas either by luminal diffusion or by interstitial leakage during gallstone impaction and pancreatitis is associated with impaired Ca(2+) signaling, we examined the effect of bile acids on pancreatic acinar cell signaling and the associated intracellular events. METHODS: Rat pancreatic acinar cells were isolated by collagenase digestion and the effects of bile acids on [Ca(2+)](i) signaling, cell survival, inflammatory signals, and the molecular and functional expressions of bile uptake transporters were analyzed. RESULTS: Bile acids specifically inhibited the sarco/endoplasmic reticulum Ca(2+) ATPase pump to chronically deplete part of the Ca(2+) stored in the endoplasmic reticulum. This in turn led to the activation of capacitative Ca(2+) entry and a chronic [Ca(2+)](i) load. The increase in [Ca(2+)](i) and Ca(2+) load activated the inflammation-associated signals of c-Jun amino-terminal kinases and NF-kappaB and led to cell death, which was inhibited by buffering [Ca(2+)](i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid. A comprehensive molecular analysis of bile acid transporters revealed that pancreatic acinar cells express the bile uptake transporters Na(+)-taurocholate co-transporting polypeptide and organic anion transporting polypeptide in the luminal and basolateral membranes, respectively. Bile acid uptake into acinar cells was in part Na(+)-dependent and in part Na(+)-independent, suggesting that both transporters contribute to bile acid influx into acinar cells. CONCLUSIONS: These results suggest that bile acids can be transported into pancreatic acinar cells through specific membrane transporters and induce cell death by impairing cellular Ca(2+) signaling.     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

Inversion of neurovascular coupling by subarachnoid blood depends on large-conductance Ca2+-activated K+ (BK) channels

The cellular events that cause ischemic neurological damage following aneurysmal subarachnoid hemorrhage (SAH) have remained elusive. We report that subarachnoid blood profoundly impacts communication within the neurovascular unit-neurons, astrocytes, and arterioles-causing inversion of neurovascular coupling. Elevation of astrocytic endfoot Ca(2+) to ～400 nM by neuronal stimulation or to ～300 nM by Ca(2+) uncaging dilated parenchymal arterioles in control brain slices but caused vasoconstriction in post-SAH brain slices. Inhibition of K(+) efflux via astrocytic endfoot large-conductance Ca(2+)-activated K(+) (BK) channels prevented both neurally evoked vasodilation (control) and vasoconstriction (SAH). Consistent with the dual vasodilator/vasoconstrictor action of extracellular K(+) ([K(+)](o)), [K(+)](o) <10 mM dilated and [K(+)](o) >20 mM constricted isolated brain cortex parenchymal arterioles with or without SAH. Notably, elevation of external K(+) to 10 mM caused vasodilation in brain slices from control animals but caused a modest constriction in brain slices from SAH model rats; this latter effect was reversed by BK channel inhibition, which restored K(+)-induced dilations. Importantly, the amplitude of spontaneous astrocytic Ca(2+) oscillations was increased after SAH, with peak Ca(2+) reaching ～490 nM. Our data support a model in which SAH increases the amplitude of spontaneous astrocytic Ca(2+) oscillations sufficiently to activate endfoot BK channels and elevate [K(+)](o) in the restricted perivascular space. Abnormally elevated basal [K(+)](o) combined with further K(+) efflux stimulated by neuronal activity elevates [K(+)](o) above the dilation/constriction threshold, switching the polarity of arteriolar responses to vasoconstriction. Inversion of neurovascular coupling may contribute to the decreased cerebral blood flow and development of neurological deficits that commonly follow SAH.     

In situ Ca2+ dynamics of Purkinje fibers and its interconnection with subjacent ventricular myocytes

Purkinje fibers play essential roles in impulse propagation to the ventricles, and their functional impairment can become arrhythmogenic. However, little is known about precise spatiotemporal pattern(s) of interconnection between Purkinje-fiber network and the underlying ventricular myocardium within the heart. To address this issue, we simultaneously visualized intracellular Ca(2+) dynamics at Purkinje fibers and subjacent ventricular myocytes in Langendorff-perfused rat hearts using multi-pinhole type, rapid-scanning confocal microscopy. Under recording of electrocardiogram at room temperature spatiotemporal changes in fluo3-fluorescence intensity were visualized on the subendocardial region of the right-ventricular septum. Staining of the heart with either fluo3, acetylthiocholine iodide (ATCHI), or di-4-ANEPPS revealed characteristic structures of Purkinje fibers. During sinus rhythm (about 60 bpm) or atrial pacing (up to 3 Hz) each Purkinje-fiber exhibited spatiotemporally synchronous Ca(2+) transients nearly simultaneously to ventricular excitation. Ca(2+) transients in individual fibers were still synchronized within the Purkinje-fiber network not only under high-K(+) (8 mM) perfusion-induced Purkinje-to-ventricular (P-V) conduction delay, but also under unidirectional, orthodromic P-V block produced by 10-mM K(+) perfusion. While spontaneous, asynchronous intracellular Ca(2+) waves were identified in injured fibers of Purkinje network locally, surrounding fibers still exhibited Ca(2+) transients synchronously to ventricular excitation. In summary, these results are the first demonstration of intracellular Ca(2+) dynamics in the Purkinje-fiber network in situ. The synchronous Ca(2+) transients, preserved even under P-V conduction disturbances or under emergence of Ca(2+) waves, imply a syncytial role of Purkinje fibers as a specialized conduction system, whereas unidirectional block at P-V junctions indicates a substrate for reentrant arrhythmias.     

Exchange protein activated by cAMP (Epac) mediates cAMP activation of p38 MAPK and modulation of Ca2+-dependent K+ channels in cerebellar neurons

The exchange factor directly activated by cAMP (Epac) is a newly discovered direct target for cAMP and a guanine-nucleotide exchange factor for the small GTPase Rap. Little is known about the neuronal functions of Epac. Here we show that activation of Epac by specific cAMP analogs or by the pituitary adenylate cyclase-activating polypeptide induces a potent activation of the Ca2+-sensitive big K+ channel, slight membrane hyperpolarization, and increased after-hyperpolarization in cultured cerebellar granule cells. These effects involve activation of Rap and p38 MAPK, which mobilizes intracellular Ca2+ stores. These findings reveal a cAMP Epac-dependent and protein kinase A-independent signaling cascade that controls neuronal excitability.     

2-iodomelatonin prevents apoptosis of cerebellar granule neurons via inhibition of A-type transient outward K+ currents

Compelling evidence indicates that excessive K+ efflux and intracellular K+ depletion are key early steps in apoptosis. Previously, we reported that apoptosis of cerebellar granular neurons induced by incubation under low K+ (5 mM) conditions was associated with an increase in delayed rectifier outward K+ current (IK) amplitude and caspase-3 activity. Moreover, the melatonin receptor antagonist 4P-PDOT abrogated the effects of 2-iodomelatonin on IK augmentation, caspase-3 activity and apoptosis. Here, we show that incubation under low K+/serum-free conditions for 6 hr led to a dramatic increase in the A-type transient outward K+ current (IA) (a 27% increase; n=31); in addition, fluorescence staining showed that under these conditions, cell viability decreased by 30% compared with the control. Treatment with 2-iodomelatonin inhibited the IA amplitude recorded from control and apoptotic cells in a concentration-dependent manner and modified the IA channel activation kinetics of cells under control conditions. Moreover, 2-iodomelatonin increased the viability of cell undergoing apoptosis. Interestingly, 4P-PDOT did not abrogate the effect of 2-iodomelatonin on IA augmentation under these conditions; in the presence of 4P-PDOT (100 microm), 2-iodomelatonin reduced the average IA by 41+/-4%, which was similar to the effect of 2-iodomelatonin alone. These results suggest that the neuroprotective effects of 2-idomelatonin are not only because of its antioxidant or receptor-activating properties, but rather that 2-iodomelatonin may inhibit IA channels by acting as a channel blocker.     

Ca2+-activated K+ channels from an insulin-secreting cell line incorporated into planar lipid bilayers

Summary This study evaluates the use of the planar lipid bilayer as a functional assay of Ca²⁺-activated K⁺ channel activity for use in purification of the channel protein. Ca²⁺-activated K⁺ channels from the plasma membrane of an insulin-secreting hamster Beta-cell line (HIT T15) were incorporated into planar lipid bilayers. The single channel conductance was 233 picoSiemens (pS) in symmetrical 140 mmol/l KCl and the channel was strongly K⁺-selective (p_Cl/p_K=0.046; P_Na/P_K=0.027). Channels incorporated into the bilayer with two orientations. In 65 % of cases, the probability of the channel being open was increased by raising calcium on the cis side of the bilayer (to which the membrane vesicles were added) or by making the cis side potential more positive. At a membrane potential of + 20 mV, which is close to the peak of the Beta-cell action potential, channel activity was half-maximal at a Ca²⁺ concentration of about 15 mol/l. Charybdotoxin greatly reduced the probability of the channel being open when added to the side opposite to that at which Ca²⁺ activated the channel. These results resemble those found for Ca²⁺-activated K⁺ channels in native Beta cell membranes and indicate that the channel properties are not significantly altered by incorporation in a planar lipid bilayer.     

Postsynaptic P/Q-type Ca2+ channel in Purkinje cell mediates synaptic competition and elimination in developing cerebellum

Neural circuits are initially redundant but rearranged through activity-dependent synapse elimination during postnatal development. This process is crucial for shaping mature neural circuits and for proper brain function. At birth, Purkinje cells (PCs) in the cerebellum are innervated by multiple climbing fibers (CFs) with similar synaptic strengths. During postnatal development, a single CF is selectively strengthened in each PC through synaptic competition, the strengthened single CF undergoes translocation to a PC dendrite, and massive elimination of redundant CF synapses follows. To investigate the cellular mechanisms of this activity-dependent synaptic refinement, we generated mice with PC-selective deletion of the Ca(v)2.1 P/Q-type Ca(2+) channel, the major voltage-dependent Ca(2+) channel in PCs. In the PC-selective Ca(v)2.1 knockout mice, Ca(2+) transients induced by spontaneous CF inputs are markedly reduced in PCs in vivo. Not a single but multiple CFs were equally strengthened in each PC from postnatal day 5 (P5) to P8, multiple CFs underwent translocation to PC dendrites, and subsequent synapse elimination until around P12 was severely impaired. Thus, P/Q-type Ca(2+) channels in postsynaptic PCs mediate synaptic competition among multiple CFs and trigger synapse elimination in developing cerebellum.     

Dose-dependent activation of Ca2+-activated K+ channels by ethanol contributes to improved endothelial cell functions

BACKGROUND: Regular moderate alcohol (EtOH) intake seems to protect against both coronary artery disease and ischemic stroke, whereas the risk increases with heavy EtOH consumption. Effects of EtOH on endothelial cell function may be relevant to these disparate effects. Potassium channels play an important role in the regulation of endothelial cell functions. Therefore, we investigated whether Ca-activated K channels (BKCa) are modulated by EtOH. Furthermore, we examined whether EtOH-induced changes of endothelial nitric oxide (NO) formation and cell proliferation are due to BKCa activation. METHODS: The patch-clamp technique was used to investigate BKCa activity in cultured human umbilical vein endothelial cells (HUVEC). NO formation was analyzed by using the fluorescence dye 4,5-diaminofluorescein. Endothelial proliferation was examined by using cell counts and measuring [H]thymidine incorporation. RESULTS: EtOH dose-dependently (10-150 mmol/liter) modulated BKCa-activity, with the highest increase of open-state probability at a concentration of 50 mmol/liter (n = 13; p < 0.05). Inside-out recordings revealed that this effect was due to direct BKCa activation, whereas open-state probability was not changed in cell-attached recordings after pertussis toxin preincubation. EtOH (10 and 50 mmol/liter) caused a significant increase of NO levels, which was blocked by the highly selective BKCa inhibitor iberiotoxin (100 nmol/l; n = 30; p < 0.05). Higher concentrations of EtOH (100 and 150 mmol/liter) significantly reduced NO synthesis (n = 30; p < 0.05). Both methods revealed a significant increase of HUVEC proliferation, which was inhibited by iberiotoxin (n = 30; p < 0.05). At a concentration of 150 mmol/liter, EtOH caused a significant reduction of endothelial proliferation. CONCLUSIONS: EtOH directly activates BKCa in HUVEC, leading to an increase of endothelial proliferation and production of NO. These results indicate a possible beneficial effect of low-dose EtOH on endothelial function, whereas higher concentrations must be considered as harmful.     

在人小肠上皮细胞膜上检出钙激活性钾通道(英文)

目的 :研究人类小肠上皮细胞 （Intestine40 7cell lines）膜上钙激活性钾通道的分子生物学特征及其电生理学特性。方法 :用 RT- PCR方法检测培养的人类小肠上皮细胞膜上钙激活性钾通道的表达形式 ,用膜片钳的全细胞记录及单通道记录法探讨了该通道的电生理学特点。结果 :RT- PCR证实该细胞系有 interm ediate- conductance（IK）钙激活性钾通道表达 ,而没有 large- conductance（BK）和 small- conductance（SK）钙激活性钾通道的表达。电生理学研究表明 ,Ionom ycin引导的全细胞电流显示该钙激活性钾电流具有内向整流性并可被特异度 IK钙激活性钾通道阻断剂 clotrimazole所抑制 ,但特异度 SK钙激活性钾通道阻断剂 apam in对该电流无明显抑制作用。单通道记录法证实该通道的电导为 30± 2 p S,通道活性对细胞内 Ca2 +浓度有明显依赖性。结论 :分子生物学和电生理研究证实了人类小肠上皮细胞膜上有 IK钙激活性钾通道 ,其生理学意义有待进一步研究     

Products of tryptophan catabolism induce Ca2+ release and modulate the cell cycle of Plasmodium falciparum malaria parasites

Intraerythrocytic malaria parasites develop in a highly synchronous manner. We have previously shown that the host hormone melatonin regulates the circadian rhythm of the rodent malaria parasite, Plasmodium chabaudi, through a Ca2+-based mechanism. Here we show that melatonin and other molecules derived from tryptophan, i.e. N-acetylserotonin, serotonin and tryptamine, also modulate the cell cycle of human malaria parasite P. falciparum by inducing an increase in cytosolic free Ca2+. This occurs independently of the extracellular Ca2+ concentration, indicating that these molecules induce Ca2+ mobilization from intracellular stores in the trophozoite. This in turn leads to an increase in the proportion of schizonts. The effects of the indolamines in increasing cytosolic free Ca2+ and modulating the parasite cell cycle are both abrogated by an antagonist of the melatonin receptor, luzindole, and by the phospholipase inhibitor, U73122.     

Effect of ageing on Ca2+ uptake via NMDA receptors into barrel cortex slices of spontaneously hypertensive rats

In the normal ageing cortex of the brain there is a group of dying neurons with shrinking dendritic trees and a group of surviving neurons with expanding dendritic trees. The ageing process affects neurotransmitter systems, including glutamate neurons and NMDA receptors. Calcium is an important signaling molecule. It enters brain cells through NMDA receptors and voltage-gated calcium channels. Since NMDA receptors play an important role in brain plasticity, calcium uptake through NMDA receptors can be used as a measure of brain activity. This study therefore sought to determine the effect of ageing on NMDA-stimulated Ca²⁺ uptake into barrel cortex slices of Spontaneously Hypertensive Rats (SHR) compared to control Wistar-Kyoto rats (WKY). Young rats (prepuberty, 4–6 weeks) and adult rats (14–16 weeks) were used in the study. The results show a significant decrease in NMDA-stimulated Ca²⁺ uptake in adult rats compared to their young litter-mates. It can be concluded that ageing negatively affects NMDA-stimulated Ca²⁺ uptake into barrel cortex slices of SHR and WKY.     

高血压大鼠冠状动脉平滑肌细胞大电导钙激活钾通道的变化

目的研究在高血压背景下大电导钙激活钾(BK)通道的功能改变及其机制。方法用酶解消化方法分离12～16周龄自发性高血压大鼠(SHR)和WKY大鼠冠状动脉平滑肌细胞(CASMCS),采用膜片钳全细胞模式记录SHR和WKY大鼠CASMCSBK电流,SHR和WKY大鼠BKα亚基和β1亚基mRNA水平用实时定量取聚合酶链式反应和凝胶电泳测定,蛋白水平表达用免疫组化的方法测定。结果 SHR大鼠CASMCS(n=6)BK电流密度比WKY(n=7)高2.03±0.62(P0.01);在mRNA水平,BKβ1亚基表达SHR组明显高于WKY组5.534±1.03倍(n=4,P0.05),BKα亚基表达则无明显差异(1.266±0.12,n=4,P0.05);BKβ1亚基和α亚基的表达SHR大鼠高于WKY大鼠。结论 SHR大鼠CASMCSBK电流密度比WKY大鼠增大,在mRNA和蛋白水平上BKβ1亚基表达也比WKY大鼠增强,这种变化可能是机体在高血压时自我调节的结果 。     

Energy coupling in liver mitochondria from dystrophic mice: differential sensitivity of oxidative phosphorylation and Ca2+ uptake to K+.

Studies were carried out to examine oxidative phosphorylation, cation uptake, and electrokinetic properties of liver mitochondria from genetically dystrophic mice in comparison with those from livers of littermate controls. While no differences were seen with respect to the rates of substrate oxidation, ADP/oxygen ratio, and RCl and cytochrome content, the mitochondria from the dystropic group were characterized by an elevated basal ATPase activity in the presence of NaCl. Additionally, these mitochondria were highly sensitive to high concentrations of exogenously added K+ that, besides stimulating state 4 respiration, caused uncoupling in the mitochondria. These mitochondria accumulated Ca2+ at a higher rate, and unlike the controls, Ca2+ uptake was not sensitive to exogenously added K+. It was also observed that the net negative charge on mitochondria decreased significantly in the dystrophic state. It is thus apparent that muscular dystrophy manifests itself also in terms of alteration in the membrane properties of liver mitochondria.     

17beta-estradiol at physiological concentrations augments Ca(2+) -activated K+ currents via estrogen receptor beta in the gonadotropin-releasing hormone neuronal cell line GT1-7

Estrogens play essential roles in the neuroendocrine control of reproduction. In the present study, we focused on the effects of 17beta-estradiol (E2) on the K(+) currents that regulate neuronal cell excitability and carried out perforated patch-clamp experiments with the GnRH-secreting neuronal cell line GT1-7. We revealed that a 3-d incubation with E2 at physiological concentrations (100 pm to 1 nm) augmented Ca(2+)-activated K(+) [K(Ca)] currents without influencing Ca(2+)-insensitive voltage-gated K(+) currents in GT1-7 cells. Acute application of E2 (1 nm) had no effect on the either type of K(+) current. The augmentation was completely blocked by an estrogen receptor (ER) antagonist, ICI-182,780. An ERbeta-selective agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile, augmented the K(Ca) currents, although an ERalpha-selective agonist, 4,4',4'-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol, had no effect. Knockdown of ERbeta by means of RNA interference blocked the effect of E2 on the K(Ca) currents. Furthermore, semiquantitative RT-PCR analysis revealed that the levels of BK channel subunit mRNAs for alpha and beta4 were significantly increased by incubating cells with 300 pm E2 for 3 d. In conclusion, E2 at physiological concentrations augments K(Ca) currents through ERbeta in the GT1-7 GnRH neuronal cell line and increases the expression of the BK channel subunit mRNAs, alpha and beta4.     

Cholangiocyte cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP signaling

BACKGROUND & AIMS: Cholangiocytes have primary cilia extending from the apical plasma membrane into the ductal lumen. While the physiologic significance of cholangiocyte cilia is unknown, studies in renal epithelia suggest that primary cilia possess sensory functions. Here, we tested the hypothesis that cholangiocyte cilia are sensory organelles that detect and transmit luminal bile flow stimuli into intracellular Ca2+ ([Ca2+]i) and adenosine 3',5'-cyclic monophosphate (cAMP) signaling. METHODS: Scanning electron microscopy, transmission electron microscopy, and immunofluorescent confocal microscopy of rat isolated intrahepatic bile duct units (IBDUs) were used to detect and characterize cholangiocyte cilia. The fluid flow-induced changes in Ca2+ and cAMP levels in cholangiocytes of microperfused IBDUs were detected by epifluorescence microscopy and a fluorescence assay, respectively. RESULTS: In microperfused IBDUs, luminal fluid flow induced an increase in [Ca2+]i and caused suppression of the forskolin-stimulated cAMP increase. The fluid flow-induced changes in [Ca2+]i and cAMP levels were significantly reduced or abolished when cilia were removed by chloral hydrate or when ciliary-associated proteins polycystin-1 (a mechanoreceptor), polycystin-2 (a Ca2+ channel), and the Ca2+-inhibitable adenylyl cyclase isoform 6 were individually down-regulated by small interfering RNAs. CONCLUSIONS: Cholangiocyte cilia are sensory organelles containing polycystin-1, polycystin-2, and adenylyl cyclase isoform 6 through which luminal fluid flow affects both [Ca2+]i and cAMP signaling in the cell. The data suggest a new model for regulation of ductal bile secretion involving cholangiocyte cilia.