Role of K+ channels in EDHF-dependent relaxation induced by acetylcholine in canine coronary artery

Summary To identify the K⁺ channels responsible for endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation, we studied the effects of various K⁺ channel blockers on acetylcholine-induced relaxation, which persists even in the presence of both an inhibitor of nitric oxide synthase and that of cyclooxygenase, in canine coronary artery rings. A nonselective K⁺ channel blocker, tetrabutylammonium (TBA), a large and intermediate conductance Ca²⁺-activated K⁺ channel blocker, charybdotoxin (CTX), and a voltage-dependent K⁺ channel blocker, 4-aminopyridine (4-AP), significantly inhibited this residual relaxation. A combined treatment with CTX and 4-AP almost completely blocked the relaxation. Neither a large (iberiotoxin) nor a small (apamin) conductance Ca²⁺-activated K⁺ channel blocker blocked the relaxation. We also investigated effects of K⁺ channel blockers on basal tone to determine whether or not EDHF is involved in regulating basal tone. TBA and CTX substantially raised basal tone to a greater degree in endothelium-intact preparations than in endothelium-denuded preparations. These results indicate that EDHF may exert its relaxing action through intermediate conductance Ca²⁺-activated and voltage-dependent K⁺ channels in canine coronary arteries. In addition, EDHF may play a role in maintaining basal vascular tone. This study was supported in part by a Grant-in-Aid for Scientific Research (B07457167) from the Ministry of Education, Science and Culture of Japan.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Ca2+-activated K+ channels in human smooth muscle cells of coronary atherosclerotic plaques and coronary media segments

The behavior of Ca²⁺-activated K⁺ channels of large conductance (BK_Ca) in smooth muscle cells, which were obtained from atherosclerotic plaque material (SMCP) and from media segments (SMCM) of human coronary arteries, were compared using the patch-clamp technique. Voltage-clamp protocols in cell-attached patches revealed the characteristic voltage-dependent activation of BK_Ca in both cell groups. Single-channel conduction was 216.4±16.7pS (n=6) in SMCP and 199.9±6.7pS (n=6) in SMCM in symmetrical 140 mMK⁺ solutions. Using outside-out patches, external perfusion with 500 M tetraethylammonium ions caused a typical flickery block of the unitary current. The selective BK_Ca channel inhibitor iberiotoxin (50 nM) effectively blocked BK_Ca channel activity. Comparing BK_Ca open-state probabilities (P₀) at +80 mV in cell-attached patches, a highly significant difference between SMCP (P₀=0.1438±0.1301; n=15) and SMCM (P₀=0.0093±0.0044; n=15; Kruskal-Wallis test, p<0.001) was found. In contrast to this finding, there was no significant difference in the open-state probability of BK_Ca between SMCP (P₀=0.0542±0.0237; n=9) and SMCM (P₀=0.0472±0.0218; n=10; p=n.s.) using inside-out patches. The results show an interesting difference in the behavior of large conductance Ca²⁺-activated K⁺ channel in SMCP compared to SMCM with a significantly higher channel activity in human smooth muscle cells obtained from coronary atherosclerotic plaque material. This finding may indicate an important functional role of BK_Ca channels in the development of atherosclerosis.     

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels

Summary Hydrogen peroxide (H₂O₂) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H₂O₂ was markedly attenuated by 10μM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100nM charybdotoxin, an inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels. A combination of the ODQ and charybdotoxin abolished the H₂O₂-induced relaxation. Pretreatment with 25 μM of an Rp stereoisomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), 20μM glibenclamide, or 1mM 4-aminopyridine did not affect the vascular response to H₂O₂. The presence of catalase at 1000U/ml significantly attenuated the H₂O₂-induced relaxation. Exposure of cultured smooth muscle cells to H₂O₂ activated K_Ca channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of K_Ca channels. Rp-cAMPS did not inhibit the H₂O₂-induced activation of K_Ca channels. The activation of K_Ca channels by H₂O₂ was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H₂O₂ activated K_Ca channels in a concentration-dependent manner. In inside-out patches, H₂O₂ significantly activated K_Ca channels through a process independent of cyclic guanosine 3′,5′-monophosphate (cGMP). In conclusion, H₂O₂ elicits vascular relaxation due to activation of K_Ca channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.     

Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

Epinephrine, through its action on β-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca²⁺ ([Ca²⁺]_i) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca²⁺ influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca²⁺ dye, it was found that epinephrine and isoprenaline, a β-adrenoceptor agonist, induced endothelial Ca²⁺ influx in the presence of bradykinin. This Ca²⁺ influx was inhibited by L-cis-diltiazem and LY-83583, and by a β₂-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca²⁺ influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca²⁺ influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate β-adrenoceptor agonist-induced endothelial Ca²⁺ influx and subsequent vascular dilation.     

Effects of adult-onset streptozotocin-induced diabetes on the rat brain antioxidant status and the activities of acetylcholinesterase, (Na+,K+)- and Mg2+-ATPase: modulation by L-cysteine

Uncontrolled diabetes is known to affect the nervous system. The aim of this study was to investigate the effect of the antioxidant L-cysteine (Cys) on the changes caused by adult-onset streptozotocin (STZ)-induced diabetes on the rat brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), (Na⁺,K⁺)-ATPase and Mg²⁺-ATPase. Thirty-eight male Wistar rats were divided into six groups: C_A (8-week-control), C_B (8-week-control + 1-week-saline-treated), C + Cys (8-week-control + 1-week-Cys-treated), D_A (8-week-diabetic), D_B (8-week-diabetic + 1-week-saline-treated) and D + Cys (8-week-diabetic + 1-week-Cys-treated). All diabetic rats were once treated with an intraperitoneal (i.p.) STZ injection (50 mg/kg body weight) at the beginning of the experiment, while all Cys-treated groups received i.p. injections of Cys 7 mg/kg body weight (daily, for 1-week, during the 9th-week). Whole rat brain parameters were measured spectrophotometrically. In vitro incubation with 0.83 mM of Cys or 10 mM of STZ for 3 h was performed on brain homogenate samples from groups C_B and D_B, in order to study the enzymes’ activities. Diabetic rats exhibited a statistically significant reduction in brain TAS (−28%, D_A vs C_A;−30%, D_B vs C_B) that was reversed after 1-week-Cys-administration into basal levels. Diabetes caused a significant increase in AChE activity (+27%, D_A vs C_A; +15%, D_B vs C_B), that was further enhanced by Cys-administration (+57%, D + Cys vs C_B). The C + Cys group exhibited no significant difference compared to the C_B group in TAS (+2%), but showed a significantly increased AChE activity (+66%, C + Cys vs C_B). Diabetic rats exhibited a significant reduction in the activity of Na⁺,K⁺-ATPase (−36%, D_A vs C_A;−48%, D_B vs C_B) that was not reversed after 1-week Cys administration. However, in vitro incubation with Cys partially reversed the diabetes-induced Na⁺,K⁺-ATPase inhibition. Mg²⁺-ATPase activity was not affected by STZ-induced diabetes, while Cys caused a significant inhibition of the enzyme, both in vivo (−14%, C + Cys vs C_B;−17%, D + Cys vs C_B) and in vitro (−16%, D_B + in vitro Cys vs C_B). In vitro incubation with STZ had no effect on the studied enzymes. The present data revealed a protective role for Cys towards the oxidative effect of diabetes on the adult rat brain. Moreover, an increase in whole brain AChE activity due to diabetes was recorded (not repeatedly established in the literature, since contradictory findings exist), that was further increased by Cys. The inhibition of Na⁺,K⁺-ATPase reflects a possible mechanism through which untreated diabetes could affect neuronal excitability, metabolic energy production and certain systems of neurotransmission. As concerns the use of Cys as a neuroprotective agent against diabetes, our in vitro findings could be indicative of a possible protective role of Cys under different in vivo experimental conditions.     

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     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Curcumin counteracts the aluminium-induced ageing-related alterations in oxidative stress, Na+, K+ ATPase and protein kinase C in adult and old rat brain regions

This study investigated the effect of curcumin on aluminium-induced alterations in ageing-related parameters: lipid peroxidation, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-s-transferase (GST), protein kinase C (PKC), Na⁺, K⁺-adenosine triphosphatase (Na⁺, K⁺-ATPase) and acetylcholinesterase (AChE) in the cerebral cortex and hippocampus of the brain of 10- and 24-month-old rats. Measurements taken from aluminium-fed rats were compared with those from rats in which curcumin and aluminium were co-administered. In aluminium-treated rats the levels of lipid peroxidation, PKC and AChE were enhanced while the activities of SOD, GPx, GST and Na⁺, K⁺-ATPase were significantly decreased in both the brain regions of both age-groups. In animals co-administered with curcumin and aluminium, the levels of lipid peroxidation, activities of PKC and AChE were significantly lowered while the activities of SOD, GPx, GST and Na⁺, K⁺-ATPase were significantly enhanced in the two brain regions studied indicating curcumin’s protective effects against aluminium toxicity. Though the magnitudes of curcumin-induced alterations varied in young and old animals, the results of the present study also demonstrated that curcumin exerts a protective effect against aluminium-induced elevation of ageing-related changes by modulating the extent of oxidative stress (by upregulating the activities of antioxidant enzymes) and by regulating the activities of Na⁺, K⁺ ATPase, PKC and AChE. Therefore, it is suggested that curcumin counters aluminium-induced enhancement in ageing-related processes.     

Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini

BACKGROUND & AIMS: The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) and the ryanodine receptor (RyR) are the principal Ca2+-release channels in cells and are believed to serve distinct roles in cytosolic Ca2+ (Ca(i)2+) signaling. This study investigated whether these receptors instead can release Ca2+ in a coordinated fashion. METHODS: Apical and basolateral Ca(i)2+ signals were monitored in rat pancreatic acinar cells by time-lapse confocal microscopy. Caged forms of second messengers were microinjected into individual cells and then photoreleased in a controlled fashion by either UV or 2-photon flash photolysis. RESULTS: InsP3 increased Ca(i)2+ primarily in the apical region of pancreatic acinar cells, whereas the RyR agonist cyclic adenosine diphosphate ribose (cADPR) increased Ca(i)2+ primarily in the basolateral region. Apical-to-basal Ca(i)2+ waves were induced by acetylcholine and initiation of these waves was blocked by the InsP3R inhibitor heparin, whereas propagation into the basolateral region was inhibited by the cADPR inhibitor 8-amino-cADPR. To examine integration of apical and basolateral Ca(i)2+ signals, Ca2+ was selectively released either apically or basolaterally using 2-photon flash photolysis. Ca(i)2+ increases were transient and localized in unstimulated cells. More complex Ca(i)2+ signaling patterns, including polarized Ca(i)2+ waves, were observed when Ca2+ was photoreleased in cells stimulated with subthreshold concentrations of acetylcholine. CONCLUSIONS: Polarized Ca(i)2+ waves are induced in acinar cells by serial activation of apical InsP3Rs and then basolateral RyRs, and subcellular release of Ca2+ coordinates the actions of these 2 types of Ca2+ channels. This subcellular integration of Ca2+-release channels shows a new level of complexity in the formation of Ca(i)2+ waves.     

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.     

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca²⁺-and voltage-activated K⁺ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged, thereby allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2⁻ cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK pore-forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate. These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.

The colonic epithelium is composed of a single layer of heterogeneous cells, covered by mucus, that separate the luminal contents from host tissues. Acting both in concert and individually, the diverse cells comprising the epithelial layer play the functions of protection (1), sensation (2, 3), transport of substances (4, 5), and repair (6). Colonic epithelial cells belong to three lineages: Absorptive enterocytes, enteroendocrine cells, and goblet cells (GCs). The colonic epithelium is morphologically organized into repeating units called crypts of Lieberkühn, where stem cells located at the base of the crypts divide and successively differentiate into the mature lineages as they migrate toward the crypt surface (7). Many of the key specialized functions of epithelial cells are, in part, defined by proteins involved in ion transport, located either on their luminal or basolateral membrane. Thus, among different gastrointestinal epithelial cells, ion channels, carriers, exchangers, and pumps work in concert to define a variety of essential functions: 1) Solute and electrolyte absorption and secretion in absorptive enterocytes (reviewed in refs. 5 and 8); 2) environment sensation and serotonin secretion by enteroendocrine cells (2, 9); and 3) mucus secretion by GCs and subsequent mucus maturation into the protective layer covering the epithelial surface (10–12). Despite this progress, ionic transport in GCs and its implications in GC physiology is a topic that remains poorly understood. Here, we address the role of the Ca²⁺- and voltage-activated K⁺ channel (BK channel) in GCs.GCs play two primary roles: One related to the maintenance of the mucosal barrier (reviewed in refs. 1 and 13) and one related with the mucosal immune homeostasis (reviewed in refs. 14 and 15). The role of GCs in barrier maintenance consists in generation of the mucus layer lining the intestinal lumen. One way GCs carry out this role is by secreting MUC2, the gel-forming mucin that forms the scaffold of the mucus layer separating luminal pathogens and commensal microbiota from the epithelial surface (11, 12, 15, 16). This separation is critical, as has been demonstrated in both animal models and humans: Mouse models with deficient mucus layer generation develop spontaneous colitis (16, 17), whereas a more penetrable mucus layer has been observed in patients with ulcerative colitis (UC), a form of human inflammatory bowel disease (IBD) (18, 19). The constant replenishment of the mucus layer involves MUC2 exocytosis from GCs, and subsequent maturation (hydration and expansion) of the secreted MUC2 to form the gel-like mucus coating the epithelium (15). Both exocytosis and maturation of MUC2 are highly dependent on anion and K⁺ transport (10–12, 20). It has been proposed that mucin exocytosis in colon requires activities of the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC1) (20, 21), and also anion and K⁺ channels whose identities are still unclear (20). It is also not clearly known whether specific ionic conductances are intrinsic to GCs or are located in the surrounding absorptive enterocytes. Although several types of K⁺ channels—including K_Ca3.1, Kv7.1, and BK channels—have been found in colonic epithelial cells (22–27), to what extent any of those K⁺ channels are specifically associated with GCs or critical to their function remains unclear. To date, most functional studies about colonic K⁺ channels have focused on their roles in electrolyte and fluid secretion/absorption of the whole colon, whereas the cellular events relating K⁺ channels to specific roles in GC function are still poorly understood.Among colonic epithelial K⁺ channels, the BK channel (also known as K_Ca1.1), the Ca²⁺- and voltage-activated K⁺ channel of high conductance, has been proposed to be the main component of colonic K⁺ secretion into the lumen (28–30). BK channels are homotetramers of the pore-forming BKα subunit, but can also contain tissue-specific regulatory subunits that critically define the functional properties of the channel (31). BK channels composed exclusively of the pore-forming BKα subunit are unlikely to be activated at the physiological conditions of epithelial cells and, as a consequence, the molecular properties of colonic BK channels that would allow them to contribute to colonic ion transport remain unclear. Recently, we established that the leucine-rich repeat-containing protein 26 (LRRC26), a BK regulatory γ-subunit, is specifically expressed in secretory epithelial cells, including GCs of the gastrointestinal tract (32). When LRRC26 is present in a BK channel complex, the resulting channel activates near normal resting physiological conditions, even in the absence of any elevation of intracellular Ca²⁺ (33).In the present study, we have specifically probed the role of BK channels in cells of the colonic epithelium and examined the impact of deletions of either the BKα subunit or LRRC26 on colonic function. Here, through recordings from identified GCs in intact colonic crypts, we show that LRRC26-associated BK channels contribute the major K⁺ current at low intracellular Ca²⁺ (∼250 nM) in mouse colonic GCs. Furthermore, the LRRC26-containing BK channels are activated near −40 mV, even in the absence of intracellular Ca²⁺. In contrast, in identified GCs from Lrrc26^−/− mice, BK current is present, but it is only activated at membrane potentials unlikely to ever occur physiologically. Surprisingly, all colonic epithelial MUC2⁻ cells sampled completely lack functional BK channels. To establish that the LRRC26-containing BK channels contribute to normal K⁺ fluxes in intact colon tissue, we show that the transepithelial current across distal colon at rest has a component dependent on LRRC26-associated BK channels, which is absent when either BKα or LRRC26 is genetically deleted. Moreover, the genetic ablation of either LRRC26 or BK channel results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate (DSS). Overall, our results suggest that normal potassium flux through LRRC26-associated BK channels in GCs has a protective role against development of colitis.     

Increase in electrically-stimulated Ca2+ release and suppression of caffeine response in diaphragm muscle of alloxan-diabetic mice compared with the denervation effect

Summary Changes in intracellular Ca²⁺ release in the diaphragm muscle of alloxan-diabetic mice were compared with changes in normal muscles and non-diabetic denervated muscles. We measured Ca²⁺ transient aequorin luminescence by direct electrical stimulation of these muscles. External Ca²⁺-free solution readily decreased the Ca²⁺ transient in normal muscles but had less of an effect in diabetic muscles. Only when the muscles were pre-injected with EGTA (reducing intracellular levels of free Ca²⁺) did the Ca²⁺ transients decrease significantly in diabetic muscles, however, there was no effect in denervated muscles. The caffeine-induced increase in Ca²⁺ transients, however, was delayed in both diabetic muscles and non-diabetic denervated muscles. The caffeine response was observed in normal muscles under the external Ca²⁺-free conditions even after EGTA-pretreatment, whereas it was suppressed, after a brief increase, in both diabetic and non-diabetic denervated muscles. These results demonstrate (1) the insensitivity of intracellular Ca²⁺ mobilization to external Ca²⁺ levels and the ready accumulation of intracellular Ca²⁺ in the cytosol in the diabetic state, (2) increased permeability to Ca²⁺ in the denervated state and (3) impairment of the Ca²⁺ pool which responds to caffeine in both diabetes and the non-diabetic denervated state. Diabetic neuromyopathy thus appears to be a state of abnormal Ca²⁺-mobilization caused secondarily by high levels of blood glucose.     

Famotidine,a histamine-2-receptor antagonist,inhibits the increase in rat gastric H+/K+-ATPase mRNA induced by intravenous infusion of gastrin 17 and histamine

We examined the effects of gastrin and histamine on rat gastric H⁺/K⁺-ATPase, the enzyme responsible for H⁺ secretion, gene expressionin vivo. Gastrin 17 (G 17) or histamine dihydrochloride (histamine) was continuously infused through the femoral vein of anesthetized rats. Gastric H⁺/K⁺-ATPase mRNA levels were measured using northern blot analysis. Infusion of G 17 and histamine increased the H⁺/K⁺-ATPase mRNA level significantly compared with basal control level or vehicle control level (P<0.01). However, pretreatment with famotidine, a potent histamine-2 (H₂)-receptor antagonist, inhibited the increase of rat gastric H⁺/K⁺-ATPase mRNA following G 17 and histamine infusion. These findings indicate that both histamine and G 17 increase expression of H⁺/K⁺-ATPase mRNA by activating H₂ receptor on the parietal cell.     

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.     

Overexpression of diacylglycerol kinase zeta inhibits endothelin-1-induced decreases in Ca2+ transients and cell shortening in mouse ventricular myocytes

Endothelin-1 (ET-1) is released in various cardiovascular disorders including congestive heart failure, and may modulate significantly the disease process by its potent action on vascular and cardiac muscle cell function and gene regulation. In adult mouse ventricular cardiomyocytes loaded with indo-1, ET-1 induced a sustained negative inotropic effect (NIE) in association with decreases in Ca²⁺ transients. The ET-1-induced effects on Ca²⁺ transients and cell shortening were abolished in diacylglycerol (DAG) kinase ζ-overexpressing mouse ventricular myocytes. A nonselective protein kinase C (PKC) inhibitor, GF109203X, inhibited the ET-1-induced decreases in Ca²⁺ transients and cell shortening in concentration-dependent manners, whereas a selective Ca²⁺-dependent PKC inhibitor, Gö6976, did not affect the ET-1-induced effects. A phospholipase Cβ inhibitor, U73122, and an inhibitor of phospholipase D, C₂-ceramide, partially, but significantly, attenuated the ET-1-induced effects. Derivatives of the respective inhibitors with no specific effects, U73343 and dihydro-C₂-ceramide, did not affect the ET-1-induced effects. Taken together, these results indicate that activation of a Ca²⁺-independent PKC isozyme by 1,2-DAG, which is generated by phospholipase Cβ and phospholipase D activation and inactivated by phosphorylation via DAG kinase, is responsible for the ET-1-induced decreases in Ca²⁺ transients and cell shortening in mouse ventricular cardiomyocytes.     

Diastolic transient inward current in long QT syndrome type 3 is caused by Ca overload and inhibited by ranolazine

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na⁺ channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca²⁺)-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized ΔKPQ LQT-3 mutation but the link to altered Ca²⁺ cycling related to mutant Na⁺ channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca²⁺ load and induction of spontaneous diastolic inward current (I_TI) in myocytes expressing ΔKPQ Na⁺ channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca²⁺, and measurement of SR Ca²⁺ content using a caffeine dump methodology. We compared the Ca²⁺ content of ΔKPQ^+/− myocytes displaying I_TI to those without spontaneous diastolic activity and found that I_TI induction correlates with higher sarcoplasmic reticulum (SR) Ca²⁺. Both spontaneous diastolic I_TI and underlying Ca²⁺ waves are inhibited by ranolazine at concentrations that preferentially target I_NaL during prolonged depolarization. Furthermore, ranolazine I_TI inhibition is accompanied by a small but significant decrease in SR Ca²⁺ content. Our results provide the first direct evidence that induction of diastolic transient inward current (I_TI) in ΔKPQ^+/− myocytes occurs under conditions of elevated SR Ca²⁺ load.