Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells

Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca²⁺]_i), known as [Ca²⁺]_i oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca²⁺]_i spikes does not. The oscillations do not persist in the absence of extracellular Ca²⁺, leading to the conclusion that entry of Ca²⁺ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl₃, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca²⁺]_i oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca²⁺]_i oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca²⁺. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca²⁺]_i oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca²⁺ entry that supports Ca²⁺ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca²⁺ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.     

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca²⁺ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca²⁺ by inhibition of SR Ca²⁺-ATPase (SERCA) led to sustained elevation of [Ca²⁺]_i that depended on extracellular Ca²⁺ and slightly enhanced Mn²⁺ entry. Enhanced background Ca²⁺ influx did not explain the raised [Ca²⁺]_i in response to SERCA inhibitors because it had marked gadolinium (Gd³⁺) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca²⁺ depletion activated SOCs. Strikingly, SOC-mediated Ca²⁺ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca²⁺]_i rise being greater than that evoked by 20 m m [K⁺]_o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca²⁺ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca²⁺ channels allowing Ca²⁺ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.     

Dissociation of the store-operated calcium current ICRAC and the Mg-nucleotide-regulated metal ion current MagNuM

Rat basophilic leukaemia cells (RBL-2H3-M1) were used to study the characteristics of the store-operated Ca²⁺ release-activated Ca²⁺ current ( I _CRAC) and the magnesium-nucleotide-regulated metal cation current (MagNuM) (which is conducted by the LTRPC7 channel). Pipette solutions containing 10 m m BAPTA and no added ATP induced both currents in the same cell, but the time to half-maximal activation for MagNuM was about two to three times slower than that of I _CRAC. Differential suppression of I _CRAC was achieved by buffering free [Ca²⁺]_i to 90 n m and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 m m Mg.ATP, 1.2 m m free [Mg²⁺]_i or 100 μ m GTP-γ-S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca²⁺ and Mg²⁺ caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg²⁺]_i, I _CRAC carried by monovalent ions inactivated more rapidly and more completely than MagNuM carried by monovalent ions. Since several studies have used divalent-free solutions on either side of the membrane to study selectivity and single-channel behaviour of I _CRAC, these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both I _CRAC and MagNuM are activated.     

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.     

Possible mechanisms of the concentration-dependent action of substance P to induce histamine release from rat peritoneal mast cells and the effect of extracellular calcium on mast-cell activation

Rat peritoneal mast cells purified on a Percoll gradient were loaded with the fluorescent Ca²⁺ indicator fura-2 and were challenged with different concentrations of substance P (SP), and intracellular calcium concentrations ([Ca²⁺]_i) were measured by a spectrofluorometric assay. SP at 5 × 10⁻⁶ mol/1 and 10⁻⁵ mol/1 caused a significant histamine release with a significant increase in [Ca²⁺]_i in a dose-dependent manner. However, SP at 10⁻⁸-10⁻⁶ mol/1 did not induce either histamine release or increase in [Ca²⁺]_i. Extracellular calcium at 0.9 mM inhibited the histamine release with a significant reduction of [Ca²⁺]ⁱ compared with that of the cells in a nominally calcium-free condition. These results indicate that the action of SP on rat mast cells relies upon [Ca²⁺]_i to induce histamine release.     

Capacitative calcium entry: from concept to molecules

Summary:  Rapid to moderately rapid changes in intracellular Ca²⁺ concentration, or Ca²⁺ signals, control a variety of critical cellular functions in the immune system. These signals are comprised of Ca²⁺ release from intracellular stores coordinated with Ca²⁺ influx across the plasma membrane. The most common mechanisms by which these two modes of signaling occur is through inositol 1,4,5-trisphosphate (IP₃)-induced release of Ca²⁺ from the endoplasmic reticulum (ER) and store-operated Ca²⁺ entry across the plasma membrane. The latter process was postulated over 20 years ago, and in just the past few years, the key molecular players have been discovered: STIM proteins serve as sensors of Ca²⁺ within the ER which communicate with and activate plasma membrane store-operated channels composed of Orai subunits. The process of store-operated Ca²⁺ entry provides support for oscillating Ca²⁺ signals from the ER and also provides direct activator Ca²⁺ that signals to a variety of downstream effectors.     

Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels

Agonist-activated Ca²⁺ signals in non-excitable cells are profoundly influenced by calcium entry via both store-operated and store-independent conductances. Recent studies have demonstrated that STIM1 plays a key role in the activation of store-operated conductances including the Ca²⁺-release-activated Ca²⁺ (CRAC) channels, and that Orai1 comprises the pore-forming component of these channels. We recently demonstrated that STIM1 also regulates the activity of the store-independent, arachidonic acid-regulated Ca²⁺ (ARC) channels, but does so in a manner entirely distinct from its regulation of the CRAC channels. This shared ability to be regulated by STIM1, together with their similar biophysical properties, suggested that these two distinct conductances may be molecularly related. Here, we report that whilst the levels of Orai1 alone determine the magnitude of the CRAC channel currents, both Orai1 and the closely related Orai3 are critical for the corresponding currents through ARC channels. Thus, in cells stably expressing STIM1, overexpression of Orai1 increases both CRAC and ARC channel currents. Whilst similar overexpression of Orai3 alone has no effect, ARC channel currents are specifically increased by expression of Orai3 in cells stably expressing Orai1. Moreover, expression of a dominant-negative mutant Orai3, either alone or in cells expressing wild-type Orai1, profoundly and specifically reduces currents through the ARC channels without affecting those through the CRAC channels, and siRNA-mediated knockdown of either Orai1 or Orai3 markedly inhibits ARC channel currents. Importantly, our data also show that the precise effects observed critically depend on which of the three proteins necessary for effective ARC channel activity (STIM1, Orai1 and Orai3) are rate limiting under the specific conditions employed.     

Discrete influx events refill depleted Ca2+ stores in a chick retinal neuron

The depletion of ER Ca²⁺ stores, following the release of Ca²⁺ during intracellular signalling, triggers the Ca²⁺ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca²⁺]_i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca²⁺ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca²⁺ entry in the form of motes.     

Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum

Unloading of endoplasmic reticulum (ER) Ca²⁺ stores activates influx of extracellular Ca²⁺ through 'store-operated' Ca²⁺ channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca²⁺ stores. Here, using high resolution imaging with the membrane-associated Ca²⁺ indicator, FFP-18, it is shown that store-operated Ca²⁺ entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane–ER junctions. In the absence of extracellular Ca²⁺, depletion of ER Ca²⁺ stores using cyclopiazonic acid, an ER Ca²⁺-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca²⁺ concentration ([Ca²⁺]_SPM) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca²⁺ causes localized Ca²⁺ influx that first increases [Ca²⁺]_SPM in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs , markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca²⁺ entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca²⁺ stores.     

Microheterogeneity of calcium signalling in dendrites

Transient changes in the intracellular concentration of free Ca²⁺ ([Ca²⁺]_i) originating from voltage- or ligand-gated influx and by ligand- or Ca²⁺-gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca²⁺ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca²⁺ pumps and Ca²⁺ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca²⁺]_i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca²⁺]_i transients with energy-dispersive X-ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca²⁺ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca²⁺ influx, but also as an intracellular Ca²⁺ source that can be mobilized by a signal cascade originating at activated synapses.     

Simultaneous imaging of Ca2+ signals in interstitial cells of Cajal and longitudinal smooth muscle cells during rhythmic activity in mouse ileum

Electrical rhythmicity in smooth muscle cells is essential for the movement of the gastrointestinal tract. Interstitial cells of Cajal (ICC) lie adjacent to smooth muscle layers and are implicated as the pacemaker cells. However, the pace making mechanism remains unclear. To study the intercellular interaction during electrical rhythm generation, we visualized changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells and myenteric ICC within segments of mouse ileum loaded with a fluorescent Ca²⁺ indicator, fluo-3. We observed rhythmic [Ca²⁺]_i changes in longitudinal smooth muscle cells travelling rapidly through the smooth muscle cell layer. Between the rhythmic Ca²⁺ transients, we found brief Ca²⁺ transients localized to small areas within smooth muscle cells. The amplitude but not the periodicity of rhythmic [Ca²⁺]_i transients in both cell types was partially inhibited by nicardipine, an L-type Ca²⁺ channel antagonist, suggesting that the rhythmic [Ca²⁺]_i transients reflect membrane potential depolarizations corresponding to both slow waves and triggered Ca²⁺ spikes. Longitudinal smooth muscle cells and myenteric ICC showed synchronous spontaneous [Ca²⁺]_i transients in eight out of 21 ileac preparations analysed. In the remaining preparations, the synchrony between ICC and smooth muscle cells was absent, although the rhythmicity of the smooth muscle cells was not disturbed. These results suggest that myenteric ICC may play multiple roles including pace making for physiological bowel movement.     

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca²⁺ release activated Ca²⁺ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca²⁺ sensor required for activation of CRAC current ( I _CRAC) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca²⁺ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I _CRAC. In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I _CRAC. The results show that cells expressing low Orai1 : STIM1 ratios produce I _CRAC with strong fast Ca²⁺-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I _CRAC with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca²⁺, Ba²⁺ and Sr²⁺ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca²⁺ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.     

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.     

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.     

Mechanisms underlying variations in excitation–contraction coupling across the mouse left ventricular free wall

Ca²⁺ release during excitation–contraction (EC) coupling varies across the left ventricular free wall. Here, we investigated the mechanisms underlying EC coupling differences between mouse left ventricular epicardial (Epi) and endocardial (Endo) myocytes. We found that diastolic and systolic [Ca²⁺]_i was higher in paced Endo than in Epi myocytes. Our data indicated that differences in action potential (AP) waveform between Epi and Endo cells only partially accounted for differences in [Ca²⁺]_i. Rather, we found that the amplitude of the [Ca²⁺]_i transient, but not its trigger – the Ca²⁺ current – was larger in Endo than in Epi cells. We also found that spontaneous Ca²⁺ spark activity was about 2.8-fold higher in Endo than in Epi cells. Interestingly, ryanodine receptor type 2 (RyR2) protein expression was nearly 2-fold higher in Endo than in Epi myocytes. Finally, we observed less Na⁺–Ca²⁺ exchanger function in Endo than in Epi cells, which was associated with decreased Ca²⁺ efflux during the AP; this contributed to higher diastolic [Ca²⁺]_i and SR Ca²⁺ in Endo than in Epi cells during pacing. We propose that transmural differences in AP waveform, SR Ca²⁺ release, and Na⁺–Ca²⁺ exchanger function underlie differences in [Ca²⁺]_i and EC coupling across the left ventricular free wall.     

Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca²⁺]_i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca²⁺]_i. With brief stimulation at LT, [Ca²⁺]_i transiently increased but returned to baseline (∼63 n m ) in < 8 min. After 15 min at LT, [Ca²⁺]_i was altered by incubating preparations in the Ca²⁺ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca²⁺]_i increased; the rate at 300 n m Ca²⁺ was ∼double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca²⁺.     

Functional role of store-operated and stretch-activated channels in murine adult skeletal muscle fibres

In skeletal muscle, Ca²⁺ is implicated in contraction, and in regulation of gene expression. An alteration of [Ca²⁺]_i homeostasis is responsible, at least partially, for the muscle degeneration that occurs after eccentric contractions in Duchenne muscular dystrophy, a disease characterized by the loss of the cytoskeletal protein dystrophin. Using patch clamp in the cell-attached configuration, we characterized the store-operated channels (SOCs) and the stretch-activated channels (SACs) present in isolated mouse skeletal muscle. SOCs were voltage independent, had a unitary conductance between 7 and 8 pS (110 m m Ca²⁺ in the pipette), and their open probability increased when the sarcoplasmic reticulum was depleted by thapsigargin. These SOCs were identical to those previously described in the pathophysiology of Duchenne muscular dystrophy. Under the same experimental conditions, we detected a channel activity that was increased by applying a negative pressure to the patch electrode. The SACs responsible for this current had the same unitary conductance and current–voltage relationship as those observed for SOCs. SOCs and SACs had a similar sensitivity to pharmacological agents such as Gd³⁺, SKF-96365, 2-aminoethoxydiphenyl borate and GsMTx4 toxin. Moreover, stimulation with IGF-1 increased the occurrence of the activity of both channel types. Together, these observations suggest that SOCs and SACs might belong to the same population or share common constituents. From a functional point of view, treatment of soleus muscle with SKF-96365 or GsMTx4 toxin increased its sensitivity to a fatigue protocol, suggesting that the influx of Ca²⁺ that occurs through these channels during contraction is also involved in force maintaining during repeated stimulations.     

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.