The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

Medical terminations of pregnancy: a viable source of tissue for cell replacement therapy for neurodegenerative disorders.

"Proof-of-principle" that cell replacement therapy works for neurodegeneration has been reported, but only using donor cells collected from fetal brain tissue obtained from surgical terminations of pregnancy. Surgical terminations of pregnancy represent an increasingly limited supply of donor cells due to the tendency towards performing medical termination in much of Europe. This imposes a severe constraint on further experimental and clinical cell transplantation research. Therefore, we explore here the feasibility of using medical termination tissue as a donor source. Products of conception were retrieved from surgical terminations over the last 7 years and from medical terminations over the last 2.5 years. The number of collections that yielded fetal tissue, viable brain tissue, and identifiable brain regions (ganglionic eminence, ventral mesencephalon, and neocortex) were recorded. We studied cell viability, cell physiological properties, and differentiation potential both in vitro and following transplantation into the central nervous system of rodent models of neurodegenerative disease. Within equivalent periods, we were able to collect substantially greater numbers of fetal remains from medical than from surgical terminations of pregnancy, and the medical terminations yielded a much higher proportion of identifiable and dissectible brain tissue. Furthermore, we demonstrate that harvested cells retain the capacity to differentiate into neurons with characteristics appropriate to the region from which they are dissected. We show that, contrary to widespread assumption, medical termination of pregnancy-derived fetal brain cells represent a feasible and more readily available source of human fetal tissue for experimental cell transplantation with the potential for use in future clinical trials in human neurodegenerative disease.

Spermine attenuates carotid body glomus cell oxygen sensing by inhibiting L-type Ca²(+) channels.

An increase in intracellular Ca²(+) is crucial to O2 sensing by the carotid body. Polyamines have been reported to modulate both the extracellular Ca²(+)-sensing receptor (CaR) and voltage-gated Ca²(+) channels in a number of cell types. Using RT-PCR and immunohistochemistry, the predominant voltage-gated Ca²(+) channels expressed in the adult rat carotid body were L (Ca(V)1.2) and N (Ca(V)2.2)-type. CaR mRNA could not be amplified from carotid bodies, but the protein was expressed in the nerve endings. Spermine inhibited the hypoxia-evoked catecholamine release from isolated carotid bodies and attenuated the depolarization- and hypoxia-evoked Ca²(+) influx into isolated glomus cells. In agreement with data from carotid body, recombinant Ca(V)1.2 was also inhibited by spermine. In contrast, the positive allosteric modulator of CaR, R-568, was without effect on hypoxia-induced catecholamine release from carotid bodies and depolarization-evoked Ca²(+) influx into glomus cells. These data show that spermine exerts a negative influence on carotid body O2 sensing by inhibiting L-type Ca²(+) channels.

Carbon monoxide is a rapid modulator of recombinant and native P2X(2) ligand-gated ion channels.

BACKGROUND AND PURPOSE: Carbon monoxide (CO) is a potent modulator of a wide variety of physiological processes, including sensory signal transduction. Many afferent sensory pathways are dependent upon purinergic neurotransmission, but direct modulation of the P2X purinoceptors by this important, endogenously produced gas has never been investigated. EXPERIMENTAL APPROACH: Whole-cell patch-clamp experiments were used to measure ATP-elicited currents in human embryonic kidney 293 cells heterologously expressing P2X(2), P2X(3), P2X(2/3) and P2X(4) receptors and in rat pheochromocytoma (PC12) cells known to express native P2X(2) receptors. Modulation was investigated using solutions containing CO gas and the CO donor molecule, tricarbonyldichlororuthenium (II) dimer (CORM-2). KEY RESULTS: CO was a potent and selective modulator of native P2X(2) receptors, and these effects were mimicked by a CO donor (CORM-2). Neither pre-incubation with 8-bromoguanosine-3',5'-cyclomonophosphate nor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (a potent blocker of soluble guanylyl cyclase) affected the ability of the CO donor to enhance the ATP-evoked P2X(2) currents. The CO donor caused a small, but significant inhibition of currents evoked by P2X(2/3) and P2X(4) receptors, but was without effect on P2X(3) receptors. CONCLUSIONS AND IMPLICATIONS: These data provided an explanation for how CO might regulate sensory neuronal traffic in physiological reflexes such as systemic oxygen sensing but also showed that CO could be used as a selective pharmacological tool to assess the involvement of homomeric P2X(2) receptors in physiological systems.

Cysteine residues in the C-terminal tail of the human BK(Ca)alpha subunit are important for channel sensitivity to carbon monoxide.

In the presence of oxygen (O(2)), carbon monoxide (CO) is synthesised from heme by endogenous hemeoxygenases, and is a powerful activator of BK(Ca) channels. This transduction pathway has been proposed to contribute to cellular O(2) sensing in rat carotid body. In the present study we have explored the role that four cysteine residues (C820, C911, C995 and C1028), located in the vicinity of the "calcium bowl" of C-terminal of human BK(Ca)-alphasubunit, have on channel CO sensitivity. Mutant BK(Ca)-alphasubunits were generated by site-directed mutagenesis (single, double and triple cysteine residue substitutions with glycine residues) and were transiently transfected into HEK 293 cells before subsequent analysis in inside-out membrane patches. Potassium cyanide (KCN) completely abolished activation of wild type BK(Ca) channels by the CO donor, tricarbonyldichlororuthenium (II) dimer, at 100microM. In the absence of KCN the CO donor increased wild-type channel activity in a concentration-dependent manner, with an EC(50) of ca. 50microM. Single cysteine point mutations of residues C820, C995 and C1028 affected neither channel characteristics nor CO EC(50) values. In contrast, the CO sensitivity of the C911G mutation was significantly decreased (EC(50) ca. 100 M). Furthermore, all double and triple mutants which contained the C911G substitution exhibited reduced CO sensitivity, whilst those which did not contain this mutation displayed essentially unaltered CO EC(50) values. These data highlight that a single cysteine residue is crucial to the activation of BK(Ca) by CO. We suggest that CO may bind to this channel subunit in a manner similar to the transition metal-dependent co-ordination which is characteristic of several enzymes, such as CO dehydrogenase.

Hydrogen sulfide inhibits human BK(Ca) channels.

Hydrogen sulfide (H(2)S) is produced endogenously in many types of mammalian cells. Evidence is now accumulating to suggest that H(2)S is an endogenous signalling molecule, with a variety of molecular targets, including ion channels. Here, we describe the effects of H(2)S on the large conductance, calcium-sensitive potassium channel (BK(Ca)). This channel contributes to carotid body glomus cell excitability and oxygen-sensitivity. The experiments were performed on HEK 293 cells, stably expressing the human BK(Ca) channel alpha subunit, using patch-clamp in the inside-out configuration. The H(2)S donor, NaSH (100microM-10 mM), inhibited BK(Ca) channels in a concentration-dependent manner with an IC(50) of ca. 670microM. In contrast to the known effects of CO donors, the H(2)S donor maximally decreased the open state probability by over 50% and shifted the half activation voltage by more than +16mV. In addition, although 1 mM KCN completely suppressed CO-evoked channel activation, it was without effect on the H(2)S-induced channel inhibition, suggesting that the effects of CO and H(2)S were non-competitive. RT-PCR showed that mRNA for both of the H(2)S-producing enzymes, cystathionine-beta-synthase and cystathionine-gamma-lyase, were expressed in HEK 293 cells and in rat carotid body. Furthermore, immunohistochemistry was able to localise cystathionine-gamma-lyase to glomus cells, indicating that the carotid body has the endogenous capacity to produce H(2)S. In conclusion, we have shown that H(2)S and CO have opposing effects on BK(Ca)channels, suggesting that these gases have separate modes of action and that they modulate carotid body activity by binding at different motifs in the BK(Ca)alphasubunit.

Effects of the polyamine spermine on arterial chemoreception.

Polyamines modulate many biological functions. Here we report a novel inhibitory modulation by spermine of catecholamine release by the rat carotid body and have identified the molecular mechanism underpinning it. We used molecular (RT-PCR and confocal microscopy) and functional (i.e., neurotransmitter release, patch clamp recording and calcium imaging) approaches to test the involvement of: (i) voltage-dependent calcium channels, and; (ii) the extracellular calcium-sensing receptor, CaR, a G protein-coupled receptor which is also activated by polyamines. RT-PCR and immunohistochemistry of isolated carotid bodies revealed that only Ca(v)1.2 and Ca(v)2.2 were expressed in type 1 cells while Ca(v)1.3, Ca(v)1.4, Ca(v)2.1, Ca(v)2.3 and Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, could not be detected. CaR expression was detected exclusively in the nerve endings. In isolated carotid bodies, the hypoxia-dependent (7% O(2) for 10 minutes) and depolarization-evoked catecholamine release were partially suppressed by pre- (and co)-incubation with 500microM spermine. In dissociated type 1 glomus cells intracellular calcium concentration did not change following spermine treatment, but this polyamine did inhibit the depolarisation-evoked calcium influx. Whole-cell patch clamp recordings of HEK293 cells stably transfected with Ca(v)1.2 demonstrated that spermine inhibits this calcium channel. Interestingly, this inhibition was not apparent if the extracellular solution contained a concentration of Ba(2) above 2 mM as the charge carrier. In conclusion, spermine attenuates catecholamine release by the carotid body principally via inhibition of Ca(v)1.2. This mechanism may represent a negative feedback, which limits transmitter release during hypoxia.

Hypoxic regulation of Ca2+ signalling in astrocytes and endothelial cells.

Acute hypoxia is well known to modulate plasmalemmal ion channels in specific tissue types, thereby modulating [Ca2+]i. Alternative mechanisms by which acute hypoxia could modulate [Ca2+]i are less well explored, particularly in non-excitable cells. Here, we describe experiments employing microfluorimetric recordings from Fura-2-loaded rat cortical astrocytes and human saphenous vein endothelial cells designed to explore any effects of hypoxia (pO2 20-30 mmHg) on [Ca2+]i. In both cell types, hypoxia evoked small rises of [Ca2+]i in the majority of cells during perfusion with a Ca(2+)-free solution, indicating hypoxia can release Ca2+ from an intracellular pool. Capacitative Ca2+ entry was observed when Ca2+ was subsequently restored to the extracellular solution. These effects were abolished by pre-treatment of cells with thapsigargin or prior application of inositol 1,4,5-trisphosphate (IP3)-generating agonists. Antioxidants fully prevented this effect of hypoxia in both cell types. Mitochondrial uncoupling significantly enhanced the effects of hypoxia in astrocytes, yet markedly suppressed the effects of hypoxia in endothelial cells. Our findings indicate that hypoxia can modulate [Ca2+]i in non-excitable cells; most importantly, it can evoke Ca2+ release from intracellular stores via a mechanism which involves reactive oxygen species. The involvement of mitochondria in this effect appears to be tissue specific.

Structural requirements for O2 sensing by the human tandem-P domain channel, hTREK1.

TREK1 is a member of the tandem-P domain K+ channel family which is expressed almost exclusively in the nervous system. It is modulated by a number of important factors including arachidonic acid and cell swelling. Since both factors are associated with brain ischemia, it has been suggested that activation of TREK1 may confer neuroprotection. However, it has been reported that the stably expressed human homologue of TREK1 is inhibited by hypoxia, calling into question its neuroprotective role in ischemia. Here, using transient transfection of HEK 293 cells with several hTREK1 mutations and whole-cell patch-clamp, we show that: hypoxic inhibition: (a) requires the C-terminal domain of the channel; (b) does not involve redox modulation of the C-terminal domain cysteine residues C365 and C399; and (c) is critically dependent on the glutamate residue at position 306. These data suggest strongly that neuroprotection is unlikely to be provided by this channel in low O2 environments and continue to cast a shadow of doubt over the precise role that TREK may have during hypoxic episodes.

Selective modulation of ligand-gated P2X purinoceptor channels by acute hypoxia is mediated by reactive oxygen species.

Purinergic excitatory synapses use ATP to mediate fast synaptic transmission via activation of P2X receptor cation channels, and this response can be altered by acute hypoxia. This study examined the effect of acute hypoxia on cloned homo- and heteromeric P2X2 and P2X3 receptors expressed in human embryonic kidney 293 cells. In cells expressing homomeric P2X2 receptors, perfusion of 5 microM ATP (EC25) induced an inward whole-cell current that showed little desensitization during repeated exposures under continuously normoxic conditions. Exposure to a hypoxic ATP solution (pO2, 25-40 mm Hg) significantly reduced the whole-cell current to 49% of normoxic control. This hypoxic inhibition of P2X2-mediated inward current was maintained across all potentials when a voltage-step protocol was applied. In contrast, currents mediated by homomeric P2X3 receptors or heteromeric P2X(2/3) receptors were insensitive to an acute hypoxic challenge. One mechanism whereby hypoxia may modulate P2X2 channels is via the production of reactive oxygen species (ROS). H2O2 (1.8 mM) reversibly reduced homomeric P2X2 whole-cell currents to 38% of control. Furthermore, H2O2 attenuated the effect of hypoxia on homomeric P2X2 whole-cell currents. Inhibitors of the mitochondrial electron transport chain that reduce (rotenone and myxothiazol) or increase (antimycin A) the production of ROS altered the magnitude of P2X2-mediated currents. In summary, this is the first report indicating that acute hypoxia is able to regulate the activity of any ligand-gated ion channel. Furthermore, our data show that acute hypoxia selectively modulates the P2X2 receptor and that the response of P2X2 receptor subunits to hypoxia is mediated through the mitochondrial production of ROS.

Regulation of recombinant human brain tandem P domain K+ channels by hypoxia: a role for O2 in the control of neuronal excitability?

The tandem P domain potassium channels, TREK1 and TASK1, are expressed throughout the brain but expression patterns do not significantly overlap. Since normal pO2 in central nervous tissue is as low as 20 mmHg and can decrease even further in ischemic disease, it is important that the behaviour of human brain ion channels is studied under conditions of acute and chronic hypoxia. This is especially true for brain-expressed tandem P-domain channels principally because they are important contributors to neuronal resting membrane potential and excitability. Here, we discuss some recent data derived from two recombinant tandem P-domain potassium channels, hTREK1 and hTASK1. Hypoxia represents a potent inhibitory influence on both channel types and occludes the activation by arachidonic acid, intracellular acidosis and membrane deformation of TREK1. This casts doubt on the idea that TREK1 activation during brain ischemia might facilitate neuroprotection via hyperpolarising neurons in which it is expressed. Interestingly, hypoxia is unable to regulate alkalotic inhibition of TREK1 suggesting that this channel may be more intimately involved in control of excitability during physiological or pathological alkalosis.

Acute hypoxia occludes hTREK-1 modulation: re-evaluation of the potential role of tandem P domain K+ channels in central neuroprotection.

The human tandem P domain K+ channel hTREK-1 (KCNK2) is distributed widely through the CNS. Here, whole-cell patch clamp recordings were employed to investigate the effects of hypoxia on hTREK-1 channels stably expressed in human embryonic kidney cells. Acute hypoxia caused a rapid and reversible inhibition of whole-cell K+ current amplitudes; this was PO2 dependent with a maximal inhibition achieved at 60 mmHg and below. In accordance with previous studies, hTREK-1 current amplitudes were enhanced by arachidonic acid. This effect was concentration dependent, with maximal enhancement observed at a concentration of 10 microM. Membrane deformation by the crenator trinitrophenol (to mimic cell swelling) or the cup former chlorpromazine (to mimic cell shrinkage) caused robust activation and inhibition of currents, respectively. However, current augmentation by either arachidonic acid or trinitrophenol was completely prevented during hypoxia; conversely, hypoxia blunted the inhibitory action of chlorpromazine. The abilities of arachidonic acid to augment currents and of hypoxia to completely abrogate this effect were also observed in cell-attached patches. Our data indicate that hypoxia interacts with hTREK-1, and occludes its modulation by arachidonic acid and membrane deformation. These findings also suggest that the potential neuroprotective role of TREK channels, which has recently been proposed, requires reconsideration since hTREK-1 activation is unlikely when ambient PO2 is below 60 mmHg - a situation which normally pertains in the CNS even during systemic normoxia.

Hypoxia inhibits human recombinant large conductance, Ca(2+)-activated K(+) (maxi-K) channels by a mechanism which is membrane delimited and Ca(2+) sensitive.

Large conductance, Ca(2+)-activated K(+) (maxi-K ) channel activity was recorded in excised, inside-out patches from HEK 293 cells stably co-expressing the alpha- and beta-subunits of human brain maxi-K channels. At +50 mV, and in the presence of 300 nM Ca2+i, single channel activity was acutely and reversibly suppressed upon reducing P(O(2)) from 150 to > 40 mmHg by over 30 %. The hypoxia-evoked reduction in current was due predominantly to suppression in NP(o), although a minor component was attributable to reduced unitary conductance of 8-12 %. Hypoxia caused an approximate doubling of the time constant for activation but was without effect on deactivation. At lower levels of Ca2+i(30 and 100 nM), hypoxic inhibition did not reach significance. In contrast, 300 nM and 1 microM Ca2+i both sustained significant hypoxic suppression of activity over the entire activating voltage range. At these two Ca2+i levels, hypoxia evoked a positive shift in the activating voltage (by approximately 10 mV at 300 nM and approximately 25 mV at 1 microM). At saturating [Ca(2+)](i) (100 microM), hypoxic inhibition was absent. Distinguishing between hypoxia-evoked changes in voltage- and/or Ca2+i-sensitivity was achieved by evoking maximal channel activity using high depolarising potentials (up to +200 mV) in the presence of 300 nM or 100 microM Ca2+i or in its virtual absence (> 1 nM). Under these experimental conditions, hypoxia caused significant channel inhibition only in the presence of 300 nM Ca2+i. Thus, since regulation was observed in excised patches, maxi-K channel inhibition by hypoxia does not require soluble intracellular components and, mechanistically, is voltage independent and Ca2+i sensitive.

Chronic hypoxia remodels voltage-gated Ca2+ entry in a human airway chemoreceptor cell line.

Arterial and airway chemoreceptors respond to acute hypoxia by depolarizing, thereby activating voltage-gated Ca2+ channels and so permitting Ca2+ entry to trigger transmitter release. Following periods of prolonged hypoxia, these cells undergo a form of remodelling which involves altered expression of ion channels. Here, we use microspectrofluorimetric recordings of voltage-gated Ca2+ entry (activated by exposure of cells to 50 mM K+) to show that chronic hypoxia suppresses such Ca2+ entry in model airway chemoreceptor (H146) cells. Furthermore, Ca2+ entry via L-type channels is suppressed, whilst entry via N-type channels is greatly enhanced. The suppressed response, together with dramatic remodelling of routes available for voltage-gated Ca2+ entry, is likely to alter significantly the acute O2 sensing properties of these cells.

Acute oxygen sensing: diverse but convergent mechanisms in airway and arterial chemoreceptors.

Airway neuroepithelial bodies sense changes in inspired O2, whereas arterial O2 levels are monitored primarily by the carotid body. Both respond to hypoxia by initiating corrective cardiorespiratory reflexes, thereby optimising gas exchange in the face of a potentially deleterious O2 supply. One unifying theme underpinning chemotransduction in these tissues is K+ channel inhibition. However, the transduction components, from O2 sensor to K+ channel, display considerable tissue specificity yet result in analogous end points. Here we highlight how emerging data are contributing to a more complete understanding of O2 chemosensing at the molecular level.

Re-evaluating the Na(+) conductance of adult rat alveolar type II pneumocytes: evidence for the involvement of cGMP-activated cation channels.

1. Alveolar epithelial type II pneumocytes were isolated and purified from adult rat lung by elastase digestion and differential adhesion, and cultured in serum-free medium for approximately 2 days on glass coverslips for subsequent patch-clamp studies employing symmetrical sodium isethionate solutions. 2. Whole-cell Na(+) currents exhibited essentially linear current-voltage relationships which were mildly inhibited (by approximately 25 %) by 10 microM amiloride. In contrast, 1 mM Zn(2+) inhibited the currents by approximately 55 % with an IC(50) of approximately 134 microM and maximal blockade achieved between 5 and 10 mM. The effects of Zn(2+) and amiloride were additive, and independent of the order of blocker addition. 3. Gd(2+), Zn(2+) and La(3+) at 10 mM were all effective at rapidly, reversibly and significantly blocking the amiloride-insensitive currents by approximately 60%. in contrast, Ni(2+) was a very weak inhibitor (30 % inhibition at 10 mM). 4. Pimozide (10 microM) caused inhibition of whole-cell cation conductance by approximately 55 %. The inhibitory effect of pimozide was concentration dependent with an IC(50) of approximately 1 microM and was maximally effective between 10 and 30 microM. Sequential addition of Zn(2+) and pimozide, in either order, revealed no overlapping inhibitory effect on the amiloride-insensitive conductance, and supported the notion that the Zn(2+)- and pimozide-sensitive currents are identical. 5. The amiloride-insensitive, Zn(2+)-blockable conductance was characterised by a Na(+)/K(+) permeability ratio (P(Na)/P(K)) of 0.73 +/- 0.02. 6. 8Br-cGMP (100 microM), a membrane-permeable analogue of cGMP, evoked a robust activation of whole-cell cation conductance to 220 % of control. This activation was apparent in either the absence or the presence of 10 microM amiloride, but was completely abolished in the presence of Zn(2+). 7. These data support the in vivo and in situ observations of a substantial amiloride-resistant Na(+) conductance, demonstrate directly that cyclic nucleotide-gated non-selective cation channels are functionally expressed in alveolar epithelial type II cells, and suggest that these channels may contribute to the fluid-reabsorptive driving force in adult lung.

Fatty acid modulation and sequence identity of fetal guinea pig alveolar type II cell amiloride-sensitive Na+ channel.

Removal of fetal lung fluid at birth is crucial to survival. In vivo, a reversal in the direction of vectorial, amiloride-sensitive Na+) transport can be stimulated by ETYA, a nonmetabolizable analogue of the naturally occurring unsaturated fatty acid, arachidonate. Using the patch-clamp technique, fetal guinea pig alveolar type II pneumocyte single Na+ channel activity was robustly activated by 10 microM arachidonate, ETYA, oleate and stearate; this was unaffected by cyclooxygenase and 5'lipoxygenase inhibitors. The Na+ channel expressed in fetal guinea pig alveolar epithelial type II pneumocytes has biophysical properties compatible with species-specific coexpression of a novel variant of alphaENaC with betaENaC. gammaENaC is either not expressed in this tissue or shares very little homology with the rat and human gamma subunit. Thus, dramatic stimulation of this channel by arachidonate explains the in vivo observation of gestation-dependent reversal of fetal transepithelial driving force and may, therefore, be of physiological significance during the transition to breathing air at birth.

Recombinant hTASK1 is an O(2)-sensitive K(+) channel.

Hypoxic inhibition of background K(+) channels is crucial to O(2) sensing by chemoreceptor tissues, but direct demonstration of O(2) sensitivity by any member of this K(+) channel family is lacking. HEK293 cells were transfected with a pcDNA3.1-hTASK1 construct; expression of hTASK1 was verified using RT-PCR and immunocytochemistry. Whole-cell K(+) currents of cells stably expressing hTASK-1 were, as anticipated, extremely sensitive to extracellular pH, within the physiological range (IC(50) approximately 7.0). All cells expressing this signature pH sensitivity were acutely modulated by pO(2); reduction of pO(2) from 150 to <40 mmHg (at pH 7.4) caused rapid and reversible suppression of pH-sensitive K(+) currents. Furthermore, these two regulatory signals clearly acted at the same channel, since the magnitude of the O(2)-sensitive current was dependent on the extracellular pH. These data represent the first direct verification that hTASK1 is O(2)-sensitive and reinforce the idea that this K(+) channel is key to O(2) sensing in chemoreceptors.

NADPH oxidase does not account fully for O2-sensing in model airway chemoreceptor cells.

A key feature of O2 sensing by chemoreceptor tissues is the hypoxic inhibition of K+ channels. However, mechanisms coupling a fall of pO2 to channel closure differ between tissues: O2 regulation of K+ channels in chemoreceptive neuroepithelial bodies and their immortal counterparts, H146 cells, involves altered reactive oxygen species generation by NADPH oxidase. In contrast, this enzyme complex is not involved in O2 sensing by the carotid body and pulmonary vasculature. Here, we provide pharmacological evidence to support a role for NADPH oxidase in hypoxic inhibition of K+ currents in H146 cells. Two structurally unrelated NADPH oxidase inhibitors, diphenylene iodonium and phenylarsine oxide, suppressed hypoxic inhibition of K+ currents recorded using the patch-clamp technique. Most importantly, however, neither inhibitor fully blocked this response. Our findings provide the first evidence that multiple mechanisms may coexist within a specific cell type to account for hypoxic suppression of K+ channel activity.

Combined antisense and pharmacological approaches implicate hTASK as an airway O(2) sensing K(+) channel.

Neuroepithelial bodies act as airway oxygen sensors. The lung carcinoma line H146 is an established model for neuroepithelial body cells. Although O(2) sensing in both cells is via NADPH oxidase H(2)O(2)/free radical production and acute hypoxia promotes K(+) channel closure and cell depolarization, the identity of the K(+) channel is still controversial. However, recent data point toward the involvement of a member of the tandem P domain family of K(+) channels. Reverse transcription-polymerase chain reaction screening indicates that all known channels other than hTWIK1 and hTRAAK are expressed in H146 cells. Our detailed pharmacological characterization of the O(2)-sensitive K(+) current described herein is compatible with the involvement of hTASK1 or hTASK3 (pH dependence, tetraethylammonium and dithiothreitol insensitivity, blockade by arachidonic acid, and halothane activation). Furthermore, we have used antisense oligodeoxynucleotides directed against hTASK1 and hTASK3 to suppress almost completely the hTASK1 protein and show that these cells no longer respond to acute hypoxia; this behavior was not mirrored in liposome-only or missense-treated cells. Finally, we have used Zn(2+) treatment as a maneuver able to discriminate between these two homologues of hTASK and show that the most likely candidate channel for O(2) sensing in these cells is hTASK3.