Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia

In the lungs, the first line of defence against bacterial infection is the thin layer of airway surface liquid (ASL) lining the airway surface. The superficial airway epithelium exhibits complex regulatory pathways that blend ion transport to adjust ASL volume to maintain proper mucociliary clearance (MCC). We hypothesized that stresses generated by airflow and transmural pressures during breathing govern ASL volume by regulating the rate of epithelial ATP release. Luminal ATP, via interactions with apical membrane P2-purinoceptors, regulates the balance of active ion secretion versus absorption to maintain ASL volume at optimal levels for MCC. In this study we tested the hypothesis that cyclic compressive stress (CCS), mimicking normal tidal breathing, regulates ASL volume in airway epithelia. Polarized tracheobronchial epithelial cultures from normal and cystic fibrosis (CF) subjects responded to a range of CCS by increasing the rate of ATP release. In normal airway epithelia, the CCS-induced increase in ASL ATP concentration was sufficient to induce purinoceptor-mediated increases in ASL height and MCC, via inhibition of epithelial Na⁺-channel-mediated Na⁺ absorption and stimulation of Cl⁻ secretion through CFTR and the Ca²⁺-activated chloride channels. In contrast, static, non-oscillatory stress did not stimulate ATP release, ion transport or MCC, emphasizing the importance of rhythmic mechanical stress for airway defence. In CF airway cultures, which exhibit basal ASL depletion, CCS was partially effective, producing less ASL volume secretion than in normal cultures, but a level sufficient to restore MCC. The present data suggest that CCS may (1) regulate ASL volume in the normal lung and (2) improve clearance in the lungs of CF patients, potentially explaining the beneficial role of exercise in lung defence.     

Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl− transport in biliary epithelial cells through a PKCζ-dependent pathway

ATP in bile is a potent secretogogue, stimulating cholangiocyte Cl⁻ and fluid secretion via binding to membrane P2 receptors, though the physiological stimuli involved in biliary ATP release are unknown. The goal of the present studies was to determine the potential role of fluid flow in biliary ATP release and secretion. In both human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers, exposure to flow increased relative ATP release which was proportional to the shear stress. In parallel studies, shear was associated with an increase in [Ca²⁺]_i and membrane Cl⁻ permeability, which were both dependent on extracellular ATP and P2 receptor stimulation. Flow-stimulated ATP release was dependent on [Ca²⁺]_i, exhibited desensitization with repetitive stimulation, and was regulated by PKCζ. In conclusion, both human and rat biliary cells exhibit flow-stimulated, PKCζ-dependent, ATP release, increases in [Ca²⁺]_i and Cl⁻ secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases.     

Hypoxia-induced secretion of serotonin from intact pulmonary neuroepithelial bodies in neonatal rabbit

We examined the effects of hypoxia on the release of serotonin (5-HT) from intact neuroepithelial body cells (NEB), presumed airway chemoreceptors, in rabbit lung slices, using amperometry with carbon fibre microelectrodes. Under normoxia ( P _O2∼155 mmHg; 1 mmHg ≈133 Pa), most NEB cells did not exhibit detectable secretory activity; however, hypoxia elicited a dose-dependent ( P _O2 range 95–18 mmHg), tetrodotoxin (TTX)-sensitive stimulation of spike-like exocytotic events, indicative of vesicular amine release. High extracellular K⁺ (50 m m ) induced a secretory response similar to that elicited by severe hypoxia. Exocytosis was stimulated in normoxic NEB cells after exposure to tetraethylammonium (20 m m ) or 4-aminopyridine (2 m m ). Hypoxia-induced secretion was abolished by the non-specific Ca²⁺ channel blocker Cd²⁺ (100 μ m ). Secretion was also largely inhibited by the L-type Ca²⁺ channel blocker nifedipine (2 μ m ), but not by the N-type Ca²⁺ channel blocker ω-conotoxin GVIA (1 μ m ). The 5-HT₃ receptor blocker ICS 205 930 also inhibited secretion from NEB cells under hypoxia. These results suggest that hypoxia stimulates 5-HT secretion from intact NEBs via inhibition of K⁺ channels, augmentation of Na⁺-dependent action potentials and calcium entry through L-type Ca²⁺ channels, as well as by positive feedback activation of 5-HT₃ autoreceptors.     

Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells

The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors. Using polarized monolayers of airway epithelial Calu-3 cells, we investigated whether mucin secretion was accompanied by nucleotide release. Electron microscopic analyses of Calu-3 cells identified subapical granules that resembled goblet cell mucin granules. Real-time confocal microscopic analyses revealed that subapical granules, labelled with FM 1-43 or quinacrine, were competent for Ca²⁺-regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca²⁺-regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu-3 cells exhibited Ca²⁺-regulated apical release of ATP and UDP-glucose, a substrate of glycosylation reactions within the secretory pathway. Neither mucin secretion nor ATP release from Calu-3 cells were affected by activation or inhibition of the cystic fibrosis transmembrane conductance regulator. In SPOC1 cells, an airway goblet cell model, purinergic P2Y₂ receptor-stimulated increase of cytosolic Ca²⁺ concentration resulted in secretion of both mucins and nucleotides. Our data suggest that nucleotide release is a mechanism by which mucin-secreting goblet cells produce paracrine signals for mucin hydration within the ASL.     

Hypo-osmotic potentiation of acetylcholine-stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. An adequate hypo-osmotic stress (−40 mos m ) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose–response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from −20 mosM to −90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca²⁺]_i and CBF, as well as potentiating ACh-stimulated rises in [Ca²⁺]_i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X₄ purinergic receptor. A hypo-osmotic stress (−40 mosM) transiently increased [Ca²⁺]_i and potentiated the ACh-stimulated [Ca²⁺]_i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca²⁺-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca²⁺]_i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X₄.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Mathematical modelling of calcium wave propagation in mammalian airway epithelium: evidence for regenerative ATP release

Airway epithelium has been shown to exhibit intracellular calcium waves after mechanical stimulation. Two classes of mechanism have been proposed to explain calcium wave propagation: diffusion through gap junctions of the intracellular messenger inositol 1,4,5-trisphosphate (IP₃), and diffusion of paracrine extracellular messengers such as ATP. We have used single cell recordings of airway epithelium to parameterize a model of an airway epithelial cell. This was then incorporated into a spatial model of a cell culture where both mechanisms for calcium wave propagation are possible. It is shown that a decreasing return on the radius of Ca²⁺ wave propagation is achieved as the amount of ATP released from the stimulated cell increases. It is therefore shown that for a Ca²⁺ wave to propagate large distances, a significant fraction of the intracellular ATP pool would be required to be released. Further to this, the radial distribution of maximal calcium response from the stimulated cell does not produce the same flat profile of maximal calcium response seen in experiential studies. This suggests that an additional mechanism is important in Ca²⁺ wave propagation, such as regenerative release of ATP from cells downstream of the stimulated cell.     

Spatial Segregation and Interaction of Calcium Signalling Mechanisms in Rat Hippocampal CA1 Pyramidal Neurons

Postsynaptic [Ca²⁺]_i increases result from Ca²⁺ entry through ligand-gated channels, entry through voltage-gated channels, or release from intracellular stores. We found that these sources have distinct spatial distributions in hippocampal CA1 pyramidal neurons. Large amplitude regenerative release of Ca²⁺ from IP₃-sensitive stores in the form of Ca²⁺ waves were found almost exclusively on the thick apical shaft. Smaller release events did not extend more than 15 μm into the oblique dendrites. These synaptically activated regenerative waves initiated at points where the stimulated oblique dendrites branch from the apical shaft. In contrast, NMDA receptor-mediated increases were observed predominantly in oblique dendrites where spines are found at high density. These [Ca²⁺]_i increases were typically more than eight times larger than [Ca²⁺]_i from this source on the main aspiny apical shaft. Ca²⁺ entry through voltage-gated channels, activated by backpropagating action potentials, was detected at all dendritic locations. These mechanisms were not independent. Ca²⁺ entry through NMDA receptor channels or voltage-gated channels (as previously demonstrated) synergistically enhanced Ca²⁺ release generated by mGluR mobilization of IP₃.     

Dynamic ATP signalling and neural development

Purinergic signalling plays a major role in the function of every organ including the brain. A growing body of evidence also suggests that purinergic signalling is important in the development of the retina, cochlea and neocortex. In these three contexts release of ATP through the spontaneous gating of connexin hemichannels in cells, respectively, of the retinal pigment epithelium, Köllicker's organ, and the radial glia triggers waves of intracellular Ca²⁺ release. In the case of the developing retina and cortex, the released ATP acts to control proliferation of neuronal precursor cells, while in the cochlea it coordinates the spontaneous activity of adjacent hair cells to refine the tonotopic maps in the cochlear nucleus. Recently ATP-derived ADP signalling has been implicated at the very earliest stages of development, notably in triggering the gene expression necessary for formation of the eye. It is now timely to test the extent to which connexin hemichannel-mediated ATP release and accompanying Ca²⁺ waves contribute to all stages of development.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion

Parietal cell (PC) proton secretion via H⁺/K⁺-ATPase requires apical K⁺ recycling. A variety of K⁺ channels and transporters are expressed in the PC and the molecular nature of the apical K⁺ recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1^+/+, ^+/– and ^−/− mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1^−/− and ^+/+ mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by ∼90% in KCNQ1^−/− gastric mucosa. Application of a high K⁺ concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1^−/− mucosa. The study demonstrates that the KCNQ1 channel provides K⁺ to the extracellular K⁺ binding site of the H⁺/K⁺-ATPase during acid secretion, and no other gastric K⁺ channel can substitute for this function.     

pH dependence and inhibition by extracellular calcium of proton currents via plasmalemmal vacuolar-type H+-ATPase in murine osteoclasts

The vacuolar-type H⁺-ATPase (V-ATPase) in the plasma membrane of a variety of cells serves as an acid-secreting pathway, and its activity is closely related to cellular functions. Massive proton secretion often leads to electrolyte disturbances in the vicinity of the cell and may in turn affect the activity of the V-ATPase. We characterized, for the first time, the proton currents mediated by plasmalemmal V-ATPase in murine osteoclast-like cells and investigated its activity over a wide range of pH gradients across the membrane (ΔpH = extracellular pH – intracellular pH). The V-ATPase currents were identified as outward H⁺ currents and were dependent on ATP and sensitive to the inhibitors bafilomycin A₁ and N , N '-dicyclohexylcarbodiimide. Although H⁺ was transported uphill, the electrochemical gradient for H⁺ affected the current. The currents were increased by elevating ΔpH and depolarization, and were reduced by lowering ΔpH and hyperpolarization. Elevation of extracellular Ca²⁺ (5–40 m m ) diminished the currents in a dose-dependent manner and made the voltage dependence more marked. Extracellular Mg²⁺ mimicked the inhibition. With 40 m m Ca²⁺, the currents decreased to < 40% at 0 mV and to < 10% at about −80 mV. Increases in the intracellular Ca²⁺ (0.5–5 μ m ) did not affect the current. The data suggest that acid secretion through the plasmalemmal V-ATPase is regulated by a combination of the pH gradient, the membrane potential and the extracellular divalent cations. In osteoclasts, the activity-dependent accumulation of acids and Ca²⁺ in the closed extracellular compartment might serve as negative feedback signals for regulating the V-ATPase.     

Electrophysiological properties of BK channels in Xenopus motor nerve terminals

Single channel properties of Ca²⁺-activated K⁺ (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone–muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca²⁺ affinity of the channel ( K _d= 7.7 μ m at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca²⁺]_i from 1 μ m to 1 m m . The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at ≥+20 mV. The activation rate increased ∼10-fold in response to a [Ca²⁺]_i increase from 1 to 100 μ m , but increased only ∼2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca²⁺]_i of 100 μ m or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca²⁺ affinity and rapid Ca²⁺ binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca²⁺] at the inner membrane surface.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

Spike-independent release of ATP from Xenopus spinal neurons evoked by activation of glutamate receptors

As the release of ATP from neurons has only been directly studied in a few cases, we have used patch sniffing to examine ATP release from Xenopus spinal neurons. ATP release was detected following intracellular current injection to evoke spikes. However, spiking was not essential as both glutamate and NMDA could evoke release of ATP in the presence of TTX. Neither acetylcholine nor high K⁺ was effective at inducing ATP release in the presence of TTX. Although Cd²⁺ blocked glutamate-evoked release of ATP suggesting a dependence on Ca²⁺ entry, neither ω-conotoxin-GVIA nor nifedipine prevented ATP release. N-type and L-type channels are thus not essential for glutamate-evoked ATP release. That glutamate receptors can elicit release in the absence of spiking suggests a close physical relationship between these receptors, the Ca²⁺ channels and release sites. As the dependence of ATP release on the influx of Ca²⁺ through Ca²⁺ channel subtypes differs from that of synaptic transmitter release, ATP may be released from sites that are distinct from those of the principal transmitter. In addition to its role as a fast transmitter, ATP may thus be released as a consequence of the activation of excitatory glutamatergic synapses and act to signal information about activity patterns in the nervous system.     

Pulmonary oedema fluid induces non-α-ENaC-dependent Na+ transport and fluid absorption in the distal lung

To determine if pulmonary oedema fluid (EF) alters ion and fluid transport of distal lung epithelium (DLE), EF was collected from rats in acute heart failure. EF, but not plasma, increased amiloride-insensitive short circuit current ( I _sc) and Na⁺-K⁺ ATPase protein content and pump activity of DLE grown in primary culture. Inhibitors of  Cl⁻  transport or cGMP-gated cation channels had a significant (   P < 0.05  ), but limited ability to block the increased I _sc. EF increased amiloride-insensitive, but not amiloride-sensitive, DLE apical membrane Na⁺ conductance. The level of mRNA encoding epithelial sodium channel (ENaC) subunits was unchanged (α, β), or decreased (γ,   P < 0.05  ) in EF-exposed DLE. EF also induced an amiloride-insensitive increase in the potential difference across murine tracheal cysts. Distal lung explants from late gestation wild-type and α-ENaC-deficient fetal mice, which normally expand due to liquid secretion, decreased in size due to liquid absorption when exposed to EF. Trypsin digestion or heat treatment of EF abrogated the ability of EF to increase amiloride-insensitive I _sc in DLE and liquid absorption by distal lung explants. Thus proteins or protein-dependent factors within cardiogenic EF induce an α-ENaC-independent and amiloride-insensitive apical membrane Na⁺ conductance and liquid absorption in the distal lung.     

Somatic Exocytosis of Serotonin Mediated by L-Type Calcium Channels in Cultured Leech Neurones

We studied somatic exocytosis of serotonin and its mediation by L-type calcium (Ca²⁺) channels in cultured Retzius neurones of the leech. Exocytosis was induced by trains of impulses at different frequencies or by depolarisation with 40 m m potassium (K⁺), and was quantified by use of the fluorescent dye FM 1–43. Stimulation increased the membrane fluorescence and produced a pattern of FM 1–43 fluorescent spots of 1.28 ± 0.01 μm in diameter, provided that Ca²⁺ was present in the bathing fluid. Individual spots lost their stain during depolarisation with 40 m m K⁺. Electron micrographs showed clusters of dense core vesicles, some of which were in contact with the cell membrane. Presynaptic structures with clear vesicles were absent from the soma. The number of fluorescent spots per soma, but not their diameter or their fluorescence intensity, depended on the frequency of stimulation. Trains at 1 Hz produced 19.5 ± 5 spots per soma, 77.9 ± 13.9 spots per soma were produced at 10 Hz and 91.5 ± 16.9 spots per soma at 20 Hz. Staining patterns were similar for neurones in culture and in situ . In the presence of the L-type Ca²⁺ channel blocker nimodipine (10 μ m ), a 20 Hz train produced only 22.9 ± 6.4 spots per soma, representing a 75 % reduction compared to control cells (   P < 0.05  ). Subsequent incubation with 10 m m caffeine to induce Ca²⁺ release from intracellular stores increased the number of spots to 73.22 ± 12.5. Blockers of N-, P-, Q- or invertebrate Ca²⁺ channels did not affect somatic exocytosis. Our results suggest that somatic exocytosis by neurones shares common mechanisms with excitable endocrine cells.     

Purinergic regulation of epithelial transport

Purinergic receptors are a family of ubiquitous transmembrane receptors comprising two classes, P1 and P2 receptors, which are activated by adenosine and extracellular nucleotides (i.e. ATP, ADP, UTP and UDP), respectively. These receptors play a significant role in regulating ion transport in epithelial tissues through a variety of intracellular signalling pathways. Activation of these receptors is partially dependent on ATP (or UTP) release from cells and its subsequent metabolism, and this release can be triggered by a number of stimuli, often in the setting of cellular damage. The function of P2Y receptor stimulation is primarily via signalling through the G_q/PLC-β pathway and subsequent activation of Ca²⁺-dependent ion channels. P1 signalling is complex, with each of the four P1 receptors A₁, A_2A, A_2B, and A₃ having a unique role in different epithelial tissue types. In colonic epithelium the A_2B receptor plays a prominent role in regulating Cl⁻ and water secretion. In airway epithelium, A_2B and A₁ receptors are implicated in the control of Cl⁻ and other currents. In the renal tubular epithelium, A₁, A_2A, and A₃ receptors have all been identified as playing a role in controlling the ionic composition of the lumenal fluid. Here we discuss the intracellular signalling pathways for each of these receptors in various epithelial tissues and their roles in pathophysiological conditions such as cystic fibrosis.     

Allosteric modulation of the mouse kir6.2 channel by intracellular H+ and ATP

The ATP-sensitive K⁺ (K_ATP) channels are regulated by intracellular H⁺ in addition to ATP, ADP, and phospholipids. Here we show evidence for the interaction of H⁺ with ATP in regulating a cloned K_ATP channel, i.e. Kir6.2 expressed with and without the SUR1 subunit. Channel sensitivity to ATP decreases at acidic pH, while the pH sensitivity also drops in the presence of ATP. These effects are more evident in the presence of the SUR1 subunit. In the Kir6.2 + SUR1, the pH sensitivity is reduced by about 0.4 pH units with 100 μM ATP and 0.6 pH units with 1 m m ATP, while a decrease in pH from 7.4 to 6.8 lowers the ATP sensitivity by about fourfold. The Kir6.2 + SUR1 currents are strongly activated at pH 5.9-6.5 even in the presence of 1 m m ATP. The modulations appear to take place at His175 and Lys185 that are involved in proton and ATP sensing, respectively. Mutation of His175 completely eliminates the pH effect on the ATP sensitivity. Similarly, the K185E mutant-channel loses the ATP-dependent modulation of the pH sensitivity. Thus, allosteric modulations of the cloned K_ATP channel by ATP and H⁺ are demonstrated. Such a regulation allows protons to activate directly the K_ATP channels and release channel inhibition by intracellular ATP; the pH effect is further enhanced with a decrease in ATP concentration as seen in several pathophysiological conditions.     

External anions and cations distinguish between AMPA and kainate receptor gating mechanisms

Possible interactions between different intracellular Ca²⁺ release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca²⁺ signals. Spontaneous, local Ca²⁺ transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 μ m ) but not by the inositol 1,4,5-trisphosphate receptor (IP₃R) blocker, 2-aminoethoxydiphenyl borate (100 μ m ), identifying them as Ca²⁺ sparks. Caffeine (10 m m ) and carbachol (10 μ m ) initiated Ca²⁺ release at sites which co-localized with each other and with any Ca²⁺ spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml⁻¹) both inhibited the global cytoplasmic [Ca²⁺] transient evoked by carbachol, confirming that it was IP₃R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca²⁺ store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP₃Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca²⁺ release through co-operation between IP₃Rs and RyRs, and implicate IP₃Rs in the regulation of Ca²⁺ store content.