Coupling of CFTR-mediated anion secretion to nucleoside transporters and adenosine homeostasis in calu-3 cells

The purpose of this study was to characterize the role of adenosine-dependent regulation of anion secretion in Calu-3 cells. RT-PCR studies showed that Calu-3 cells expressed mRNA for A2A and A2B but not A1 or A3 receptors, and for hENT1, hENT2 and hCNT3 but not hCNT1 or hCNT2 nucleoside transporters. Short-circuit current measurements indicated that A2B receptors were present in both apical and basolateral membranes, whereas A2A receptors were detected only in basolateral membranes. Uptake studies demonstrated that the majority of adenosine transport was mediated by hENT1, which was localized to both apical and basolateral membranes, with a smaller hENT2-mediated component in basolateral membranes. Whole-cell current measurements showed that application of extracellular nitrobenzylmercaptopurine ribonucleoside (NBMPR), a selective inhibitor of hENT1-mediated transport, had similar effects on whole-cell currents as the application of exogenous adenosine. Inhibitors of adenosine kinase and 5'-nucleotidase increased and decreased, respectively, whole-cell currents, whereas inhibition of adenosine deaminase had no effect. Single-channel studies showed that NBMPR and adenosine kinase inhibitors activated CFTR Cl- channels. These results suggested that the equilibrative nucleoside transporters (hENT1, hENT2) together with adenosine kinase and 5'-nucleotidase play a crucial role in the regulation of CFTR through an adenosine-dependent pathway in human airway epithelia.     

Properties of ATP-dependent K(+) channels in adrenocortical cells

Bovine adrenocortical zona fasciculata (AZF)cells express a novel ATP-dependent K⁺-permeable channel(I_AC). Whole cell and single-channel recordings were used to characterize I_AC channels withrespect to ionic selectivity, conductance, and modulation bynucleotides, inorganic phosphates, and angiotensin II (ANG II). Inoutside-out patch recordings, the activity of unitaryI_AC channels is enhanced by ATP in the patchpipette. These channels were K⁺ selective with nomeasurable Na⁺ or Ca²⁺ conductance. Insymmetrical K⁺ solutions with physiological concentrationsof divalent cations (M²⁺), I_ACchannels were outwardly rectifying with outward and inward chordconductances of 94.5 and 27.0 pS, respectively. In the absence ofM²⁺, conductance was nearly ohmic. Hydrolysis-resistantnucleotides including AMP-PNP and NaUTP were more potent than MgATP asactivators of whole cell I_AC currents. Inorganicpolytriphosphate (PPP_i) dramatically enhancedI_AC activity. In current-clamp recordings, nucleotides and PPP_i produced resting potentials in AZFcells that correlated with their effectiveness in activatingI_AC. ANG II (10 nM) inhibited whole cellI_AC currents when patch pipettes contained 5 mMMgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.Inhibition by ANG II was not reduced by selective kinase antagonists.These results demonstrate that I_AC is adistinctive K⁺-selective channel whose activity isincreased by nucleotide triphosphates and PPP_i.Furthermore, they suggest a model for I_AC gatingthat is controlled through a cycle of ATP binding and hydrolysis.

     

K+ channel KVLQT1 located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl- secretion

The cellular mechanism for Cl^– and K⁺ secretion in the colonic epithelium requires K⁺ channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K⁺ channel proteins K_VLQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (I_sc) and transepithelial conductance (G_t) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E₂ (PGE₂), and carbachol (CCh). HMR1556 (10 µM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 3831–3837, 2001), partially (50%) inhibited Cl^– secretory I_sc and G_t activated by PGE₂ and CCh in rat colon with an IC₅₀ of 55 nM, but in guinea pig distal colon Cl^– secretory I_sc and G_t were unaltered. EPI-activated K⁺-secretory I_sc and G_t also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K⁺ channel is not an essential component for producing Cl^– secretion. Other K⁺ channels present in the basolateral membrane presumably must also contribute directly to the K⁺ conductance necessary for K⁺ exit during activation of Cl^– secretion in the colonic mucosa. HMR1556; K⁺ secretion; epinephrine; prostaglandin E₂; cholinergic     

Adenosine stimulates depolarization and rise in cytoplasmic [Ca2+] in type I cells of rat carotid bodies

During hypoxia, the level of adenosine in the carotid bodies increases as a result of ATP catabolism and adenosine efflux via adenosine transporters. Using Ca²⁺ imaging, we found that adenosine, acting via A_2A receptors, triggered a rise in cytoplasmic [Ca²⁺] ([Ca²⁺]_i) in type I (glomus) cells of rat carotid bodies. The adenosine response could be mimicked by forskolin (but not its inactive analog), and could be abolished by the PKA inhibitor H89. Simultaneous measurements of membrane potential (perforated patch recording) and [Ca²⁺]_i showed that the adenosine-mediated [Ca²⁺]_i rise was accompanied by depolarization. Ni²⁺, a voltage-gated Ca²⁺ channel (VGCC) blocker, abolished the adenosine-mediated [Ca²⁺]_i rise. Although adenosine was reported to inhibit a 4-aminopyridine (4-AP)-sensitive K⁺ current, 4-AP failed to trigger any [Ca²⁺]_i rise, or to attenuate the adenosine response. In contrast, anandamide, an inhibitor of the TWIK-related acid-sensitive K⁺-1 (TASK-1) channels, triggered depolarization and [Ca²⁺]_i rise. The adenosine response was attenuated by anandamide but not by tetraethylammonium. Our results suggest that adenosine, acting via the adenylate cyclase and PKA pathways, inhibits the TASK-1 K⁺ channels. This leads to depolarization and activation of Ca²⁺ entry via VGCC. This excitatory action of adenosine on type I cells may contribute to the chemosensitivity of the carotid body during hypoxia. O₂ sensing; A_2A receptor; cAMP; protein kinase A; TWIK-related acid-sensitive K⁺ channel     

Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents

Cell shrinkageis an early prerequisite in programmed cell death, and cytoplasmicK⁺ is a dominant cation that controls intracellular ionhomeostasis and cell volume. Blockade of K⁺ channelsinhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K⁺ efflux throughvoltage-gated K⁺ (Kv) channels. In heart-derived H9c2cells, whole cell Kv currents (I_K(V)) wereisolated by using Ca²⁺-free extracellular (bath) solutionand including 5 mM ATP and 10 mM EGTA in the intracellular (pipette)solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), ablocker of Kv channels, reversibly reduced I_K(V)by 50-60% in H9c2 cells. The remaining currents during 4-APtreatment may be generated by K⁺ efflux through4-AP-insensitive K⁺ channels. Overexpression of ARC inheart-derived H9c2 cells significantly decreasedI_K(V), whereas treatment with staurosporine, apotent apoptosis inducer, enhanced I_K(V)in wild-type cells. The staurosporine-induced increase inI_K(V) was significantly suppressed and thestaurosporine-mediated apoptosis was markedly inhibited incells overexpressing ARC compared with cells transfected with thecontrol neomycin vector. These results suggest that theantiapoptotic effect of ARC is, in part, due to inhibition of Kvchannels in cardiomyocytes.

     

Differential effects of volatile and intravenous anesthetics on the activity of human TASK-1

Volatile anesthetics have been shown to activate various two-pore (2P) domain K⁺ (K_2P) channels such as TASK-1 and TREK-1 (TWIK-related acid-sensitive K⁺ channel), and mice deficient in these channels are resistant to halothane-induced anesthesia. Here, we investigated whether K_2P channels were also potentially important targets of intravenous anesthetics. Whole cell patch-clamp techniques were used to determine the effects of the commonly used intravenous anesthetics etomidate and propofol on the acid-sensitive K⁺ current in rat ventricular myocytes (which strongly express TASK-1) and selected human K_2P channels expressed in Xenopus laevis oocytes. In myocytes, etomidate decreased both inward rectifier K⁺ (K_ir) current (I_K1) and acid-sensitive outward K⁺ current at positive potentials, suggesting that this drug may inhibit TASK channels. Indeed, in addition to inhibiting guinea pig Kir2.1 expressed in oocytes, etomidate inhibited human TASK-1 (and TASK-3) in a concentration-dependent fashion. Propofol had no effect on human TASK-1 (or TASK-3) expressed in oocytes. Moreover, we showed that, similar to the known effect of halothane, sevoflurane and the purified R-(–)- and S-(+)-enantiomers of isoflurane, without stereoselectivity, activated human TASK-1. We conclude that intravenous and volatile anesthetics have dissimilar effects on K_2P channels. Human TASK-1 (and TASK-3) are insensitive to propofol but are inhibited by supraclinical concentrations of etomidate. In contrast, stimulatory effects of sevoflurane and enantiomeric isoflurane on human TASK-1 can be observed at clinically relevant concentrations. volatile anesthetics; etomidate; propofol; ion channels     

Modulation of hepatocellular swelling-activated K+ currents by phosphoinositide pathway-dependent protein kinase C

K⁺ channels participate in the regulatory volume decrease (RVD) accompanying hepatocellular nutrient uptake and bile formation. We recently identified KCNQ1 as a molecular candidate for a significant fraction of the hepatocellular swelling-activated K⁺ current (I_KVol). We have shown that the KCNQ1 inhibitor chromanol 293B significantly inhibited RVD-associated K⁺ flux in isolated perfused rat liver and used patch-clamp techniques to define the signaling pathway linking swelling to I_KVol activation. Patch-electrode dialysis of hepatocytes with solutions that maintain or increase phosphatidylinositol 4,5-bisphosphate (PIP₂) increased I_KVol, whereas conditions that decrease cellular PIP₂ decreased I_KVol. GTP and AlF₄^– stimulated I_KVol development, suggesting a role for G proteins and phospholipase C (PLC). Supporting this, the PLC blocker U-73122 decreased I_KVol and inhibited the stimulatory response to PIP₂ or GTP. Protein kinase C (PKC) is involved, because K⁺ current was enhanced by 1-oleoyl-2-acetyl-sn-glycerol and inhibited after chronic PKC stimulation with phorbol 12-myristate 13-acetate (PMA) or the PKC inhibitor GF 109203X. Both I_KVol and the accompanying membrane capacitance increase were blocked by cytochalasin D or GF 109203X. Acute PMA did not eliminate the cytochalasin D inhibition, suggesting that PKC-mediated I_KVol activation involves the cytoskeleton. Under isotonic conditions, a slowly developing K⁺ current similar to I_KVol was activated by PIP₂, lipid phosphatase inhibitors to counter PIP₂ depletion, a PLC-coupled ₁-adrenoceptor agonist, or PKC activators and was depressed by PKC inhibition, suggesting that hypotonicity is one of a set of stimuli that can activate I_KVol through a PIP₂/PKC-dependent pathway. The results indicate that PIP₂ indirectly activates hepatocellular KCNQ1-like channels via cytoskeletal rearrangement involving PKC activation. KCNQ1; patch clamp; phosphatidylinositol 4,5-bisphosphate; regulatory volume decrease     

Distinct K+ conductive pathways are required for Cl- and K+ secretion across distal colonic epithelium

Secretion of Cl^– and K⁺ in the colonic epithelium operates through a cellular mechanism requiring K⁺ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (I_sc)] and conductance (G_t) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE₂ and synergistically by PGE₂ and carbachol (PGE₂ + CCh). TRAM-34 at 0.5 µM, an inhibitor of K_Ca3.1 (IK, Kcnn4) K⁺ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 8151–8156, 2000), did not alter secretory I_sc or G_t in guinea pig or rat colon. The presence of K_Ca3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 µM, TRAM-34 inhibited I_sc and G_t activated by Epi (4%), PGE₂ (30%) and PGE₂ + CCh (60%). The IC₅₀ of 4.0 µM implicated involvement of K⁺ channels other than K_Ca3.1. The secretory responses augmented by the K⁺ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that K_Ca3.1 was not involved. Sensitivity of the synergistic response (PGE₂ + CCh) to a high concentration TRAM-34 supported a requirement for multiple K⁺ conductive pathways in secretion. Clofilium (100 µM), a quaternary ammonium, inhibited Cl^– secretory I_sc and G_t activated by PGE₂ (20%) but not K⁺ secretion activated by Epi. Thus Cl^– secretion activated by physiological secretagogues occurred without apparent activity of K_Ca3.1 channels but was dependent on other types of K⁺ channels sensitive to high concentrations of TRAM-34 and/or clofilium. epinephrine; prostaglandin E₂; cholinergic; Kcnn4; TRAM-34; clofilium     

A2B receptor activation promotes glycogen synthesis in astrocytes through modulation of gene expression

Adenosinehas been proposed as a key factor regulating the metabolic balancebetween energy supply and demand in the central nervous system. Becauseastrocytes represent an important cellular element in the control ofbrain energy metabolism, we investigated whether adenosine could inducelong-term changes of glycogen levels in primary cultures of mousecortical astrocytes. We observed that adenosine increased glycogencontent, up to 300%, in a time- (maximum at 8 h) andconcentration-dependent manner with an EC₅₀ of 9.69 µM.Pharmacological experiments using the broad-spectrum agonist5'-(N-ethylcarboxamido)adenosine (NECA) and specificagonists for the A₁, A_2A, and A₃receptors [N⁶-cyclopentyladenosine (CPA),CGS-21680, and IB-MECA, respectively] suggest that the effect ofadenosine is mediated through activation of the low-affinityA_2B adenosine receptor subtype. Interestingly, adenosineinduces in parallel the expression of the protein targeting to glycogen(PTG), one of the protein phosphatase-1 glycogen-targeting subunitsthat has been implicated in the control of glycogen levels in varioustissues. These results indicate that adenosine can exert long-termcontrol over glycogen levels in astrocytes and might therefore play asignificant role in physiological and/or pathological processesinvolving long-term modulation of brain energy metabolism.

     

Activation of K+ channels induces apoptosis in vascular smooth muscle cells

Intracellular K⁺ playsan important role in controlling the cytoplasmic ion homeostasis formaintaining cell volume and inhibiting apoptotic enzymes in thecytosol and nucleus. Cytoplasmic K⁺ concentration is mainlyregulated by K⁺ uptake viaNa⁺-K⁺-ATPase and K⁺ efflux throughK⁺ channels in the plasma membrane. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), a protonophorethat dissipates the H⁺ gradient across the inner membraneof mitochondria, induces apoptosis in many cell types. In ratand human pulmonary artery smooth muscle cells (PASMC), FCCP opened thelarge-conductance, voltage- and Ca²⁺-sensitiveK⁺ (maxi-K) channels, increased K⁺ currentsthrough maxi-K channels [I_K(Ca)], and inducedapoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM)decreased I_K(Ca) by blocking the sarcolemmalmaxi-K channels and inhibited the FCCP-induced apoptosis inPASMC cultured in media containing serum and growth factors.Furthermore, inhibition of K⁺ efflux by raisingextracellular K⁺ concentration from 5 to 40 mM alsoattenuated PASMC apoptosis induced by FCCP and theK⁺ ionophore valinomycin. These results suggest thatFCCP-mediated apoptosis in PASMC is partially due to anincrease of maxi-K channel activity. The resultant K⁺ lossthrough opened maxi-K channels may serve as a trigger for cellshrinkage and caspase activation, which are major characteristics ofapoptosis in pulmonary vascular smooth muscle cells.

     

Cl- secretory effects of EBIO in the rabbit conjunctival epithelium

Experiments were conducted to determine whether the Cl^– secretagogue, 1-ethyl-2-benzimidazolinone (EBIO), stimulates Cl^– transport in the rabbit conjunctival epithelium. For this study, epithelia were isolated in an Ussing-type chamber under short-circuit conditions. The effects of EBIO on the short-circuit current (I_sc) and transepithelial resistance (R_t) were measured under physiological conditions, as well as in experiments with altered electrolyte concentrations. Addition of 0.5 mM EBIO to the apical bath stimulated the control I_sc by 64% and reduced R_t by 21% (P < 0.05; paired data). Under Cl^–-free conditions, I_sc stimulation using EBIO was markedly attenuated. In the presence of an apical-to-basolateral K⁺ gradient and permeabilization of the apical membrane, the majority of the I_sc reflected the transcellular movement of K⁺ via basolateral K⁺ channels. Under these conditions, EBIO in combination with A23187 elicited nearly instantaneous 60–90% increases in I_sc that were sensitive to the calmodulin antagonist calmidazolium and the K⁺ channel blocker tetraethyl ammonium. In the presence of an apical-to-basolateral Cl^– gradient and nystatin permeabilization of the basolateral aspect, EBIO increased the Cl^–-dependent I_sc, an effect prevented by the channel blocker glibenclamide (0.3 mM). The latter compound also was used to determine the proportion of EBIO-evoked unidirectional ³⁶Cl^– fluxes in the presence of the Cl^– gradient that traversed the epithelium transcellularly. Overall, EBIO activated apical Cl^– channels and basolateral K⁺ channels (presumably those that are Ca²⁺ dependent), thereby suggesting that this compound, or related derivatives, may be suitable as topical agents to stimulate fluid transport across the tissue in individuals with lacrimal gland deficiencies. Ussing chamber; short-circuit current; electrolyte transport; chloride secretagogue; potassium conductance; 1-ethyl-2-benzimidazolinone; 1,10-phenanthroline     

Functional characterization of voltage-gated K+ channels in mouse pulmonary artery smooth muscle cells

Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated K⁺ (K_V) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. K_V currents (I_K(V)) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of I_K(V) in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell I_K(V) were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed I_K(V) inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell I_K(V) were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease I_K(V) by a similar amount (37%). Contribution of Ca²⁺-activated K⁺ (K_Ca) channels to whole cell I_K(V) was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with Ca²⁺-free solution had an effect on the whole cell I_K(V). Steady-state activation and inactivation curves revealed a window K⁺ current between –40 and –10 mV with a peak at –31.5 mV. Single-channel recordings revealed large-, intermediate-, and small-amplitude currents, with an averaged slope conductance of 119.4 ± 2.7, 79.8 ± 2.8, 46.0 ± 2.2, and 23.6 ± 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native K_V currents in mouse PASMCs. K_V channels     

V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice

Inward rectifier K⁺ channels (Kir) are a significant determinant of endothelial cell (EC) membrane potential, which plays an important role in endothelium-dependent vasodilatation. In the present study, several complementary strategies were applied to determine the Kir2 subunit composition of human aortic endothelial cells (HAECs). Expression levels of Kir2.1, Kir2.2, and Kir2.4 mRNA were similar, whereas Kir2.3 mRNA expression was significantly weaker. Western blot analysis showed clear Kir2.1 and Kir2.2 protein expression, but Kir2.3 protein was undetectable. Functional analysis of endothelial inward rectifier K⁺ current (I_K) demonstrated that 1) I_K current sensitivity to Ba²⁺ and pH were consistent with currents determined using Kir2.1 and Kir2.2 but not Kir2.3 and Kir2.4, and 2) unitary conductance distributions showed two prominent peaks corresponding to known unitary conductances of Kir2.1 and Kir2.2 channels with a ratio of 4:6. When HAECs were transfected with dominant-negative (dn)Kir2.x mutants, endogenous current was reduced 50% by dnKir2.1 and 85% by dnKir2.2, whereas no significant effect was observed with dnKir2.3 or dnKir2.4. These studies suggest that Kir2.2 and Kir2.1 are primary determinants of endogenous K⁺ conductance in HAECs under resting conditions and that Kir2.2 provides the dominant conductance in these cells. potassium channels; inward rectifier potassium channel     

Sequential and opposite regulation of two outward K(+) currents by ET-1 in cultured striatal astrocytes

In the brain,astrocytes represent a major target for endothelins (ETs), a family ofpeptides that can be released by several cell types and that havepotent and multiple effects on astrocytic functions. Four types ofK⁺ currents (I_K) were detected invarious proportions by patch-clamp recordings of cultured striatalastrocytes, including the A-type I_K, theinwardly rectifying I_K IR, theCa²⁺-dependent I_K(I_K Ca), and the delayed-rectifiedI_K (I_K DR). Variationsin the shape of current-voltage relationships were related mainly todifferences in the proportion of these currents. ET-1 was found toregulate with opposite effects the two more frequently recorded outwardK⁺ currents in striatal astrocytes. Indeed, this peptideinduced an initial activation of I_K Ca(composed of SK and BK channels) and a delayed long-lasting inhibitionof I_K DR. In current-clamp recordings, theactivation of I_K Ca correlated with a transient hyperpolarization, whereas the inhibition ofI_K DR correlated with a sustaineddepolarization. These ET-1-induced sequential changes inmembrane potential in astrocytes may be important for the regulation ofvoltage gradients in astrocytic networks and the maintenance ofK⁺ homeostasis in the brain microenvironment.

     

Protease-activated receptor regulation of Cl- secretion in Calu-3 cells requires prostaglandin release and CFTR activation

Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl^– secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (I_sc: thrombin, 21 ± 2 µA; SLIGRL, 83 ± 22 µA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca²⁺-chelating agent BAPTA-AM (50 µM) abolished the increase in I_sc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 µM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated I_sc was observed. In addition, basolateral treatment with the PGE₂ receptor antagonist AH-6809 (25 µM) significantly inhibited the effects of SLIGRL on I_sc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca²⁺-activated KCNN4 K⁺ channel, and the KCNQ1 K⁺ channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl^– conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl^– secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl^– secretion requires activation of CFTR and at least two distinct K⁺ channels located in the basolateral membrane. cystic fibrosis transmembrane conductance regulator; KCNQ1; calcium-activated potassium channels; KCNN4; cAMP     

Differential modulation of voltage-dependent K+ currents in colonic smooth muscle by oxidants

The effect of oxidants on voltage-dependent K⁺ currents was examined in mouse colonic smooth muscle cells. Exposure to either chloramine-T (Ch-T), an agent known to oxidize both cysteine and methionine residues, or the colon-specific oxidant monochloramine (NH₂Cl) completely suppressed the transient outward K⁺ current (I_to) while simultaneously enhancing the sustained delayed rectifier K⁺ current (I_dr). In contrast, the cysteine-specific oxidants hydrogen peroxide (H₂O₂) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) exhibited partial and slow suppression of I_to by inducing a shift in channel availability of -18 mV without affecting I_dr. After enhancement by NH₂Cl or Ch-T, I_dr was sensitive to 10 mM tetraethylammonium but not to other K⁺ channel blockers, suggesting that it represented activation of the resting I_dr and not a separate K⁺ conductance. Extracellular dithiothreitol (DTT) partially reversed the effect of H₂O₂ and DTNB on I_to but not the actions of NH₂Cl and Ch-T on either I_dr or I_to. Dialysis of myocytes with GSH (5 mM) or DTT (5 mM) prevented suppression of I_to by H₂O₂ and DTNB but did not alter the effects of NH₂Cl or Ch-T on either I_dr or I_to. Ch-T and NH₂Cl completely blocked I_to generated by murine K_v4.1, 4.2, and 4.3 in Xenopus oocytes, an effect not reversible by intracellular DTT. In contrast, intracellular DTT reversed the effect of H₂O₂ and DTNB on the cloned channels. These results suggest that I_to is suppressed via modification of both methionine and cysteine residues, whereas enhancement of I_dr likely results from methionine oxidation alone. colon; colitis; redox; ion channel     

Dietary cholesterol alters Na+/K+ selectivity at intracellular Na+/K+ pump sites in cardiac myocytes

A modest diet-induced increase in serum cholesterol in rabbits increases the sensitivity of the sarcolemmal Na⁺/K⁺ pump to intracellular Na⁺, whereas a large increase in cholesterol levels decreases the sensitivity to Na⁺. To examine the mechanisms, we isolated cardiac myocytes from controls and from rabbits with diet-induced increases in serum cholesterol. The myocytes were voltage clamped with the use of patch pipettes that contained osmotically balanced solutions with Na⁺ in a concentration of 10 mM and K⁺ in concentrations ([K⁺]_pip) ranging from 0 to 140 mM. There was no effect of dietary cholesterol on electrogenic Na⁺/K⁺ current (I_p) when pipette solutions were K⁺ free. A modest increase in serum cholesterol caused a [K⁺]_pip-dependent increase in I_p, whereas a large increase caused a [K⁺]_pip-dependent decrease in I_p. Modeling suggested that pump stimulation with a modest increase in serum cholesterol can be explained by a decrease in the microscopic association constant K_K describing the backward reaction E₁ + 2K⁺ E₂(K⁺)₂, whereas pump inhibition with a large increase in serum cholesterol can be explained by an increase in K_K. Because hypercholesterolemia upregulates angiotensin II receptors and because angiotensin II regulates the Na⁺/K⁺ pump in cardiac myocytes in a [K⁺]_pip-dependent manner, we blocked angiotensin synthesis or angiotensin II receptors in vivo in cholesterol-fed rabbits. This abolished cholesterol-induced pump inhibition. Because the -isoform of protein kinase C (PKC) mediates effects of angiotensin II on the pump, we included specific PKC-blocking peptide in patch pipette filling solutions. The peptide reversed cholesterol-induced pump inhibition. partial reactions; protein kinase C; angiotensin converting enzyme inhibitors; arteriosclerosis; insulin resistance     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

Quantitative Analysis of ATP-Dependent H+ Efflux and Pump Current Driven by an Electrogenic Pump in Nitellopsis obtusa

Internodal cells of Nitellopsis were made tonoplast-free byperfusion with a medium containing EGTA. Cytoplasmic concentrationsof solutes were controlled by a second perfusion with mediaof known composition. The electrogenic pump current (I_p), whichwas calculated from electrical data obtained from cells withand without ATP, was compared with the current carried by H⁺(I_H⁺) across the plasma membrane. A close correlation betweenI_p and I_H⁺ was found under various internal and external conditions.(1) I_p and I_H⁺ depended on the internal ATP and showed Michaelis-Mententype saturation curves. For I_p, K_m was 120 µM and themaximum current V_max was 15.1 mA m^–2, while for I_H⁺, K_mwas 160 µM and V_max was 16.6 mA m^–2. (2) I_p andI_H⁺ showed almost the same I_H²⁺ dependence. The Mg²⁺-dependentI_p was 19.5 mA m^–2, while the Mg²⁺-dependent I_H²⁺ was17.7 mA m^–2. (3) I_H²⁺ was maximal at an external pH of8 and decreased both in acidic and alkaline pH ranges. I_p wasnearly equal to I_H⁺ in the pH range between 8 and 5. (4) I_H⁺became maximal at an internal pH of 7.3, which is nearly thesame as the pH for maximal electrogenecity found by Mimura andTazawa (1984). All these facts support the idea proposed in our previous paper(Takeshige et al. 1985) that the electrogenic ion pump locatedin the plasma membrane of Nitellopsis is the H⁺ pump. ¹ Dedicated to Professor Dr. Erwin Bünning on the occasionof his 80th birthday. (Received June 21, 1985; Accepted December 20, 1985)     

Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigated the dependence of Na⁺ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (I_sc), transepithelial conductance (G_T), and transepithelial capacitance (C_T) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl--cyclodextrin (mCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mCD treatment. However, apical mCD application hampered the responses of I_sc and G_T to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in I_sc demonstrated that apical mCD treatment decreased the epithelial Na⁺ channel (ENaC) open probability (P_o) in the steady state as well as after activation of Na⁺ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by C_T measurements. Na⁺ transport activation by hypotonicity was abolished during basolateral mCD treatment as a result of reduced Na⁺/K⁺ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na⁺/K⁺ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na⁺ transport by reducing ENaC P_o. epithelial Na⁺ channel; Na⁺-K⁺-ATPase activity; short-circuit current; methyl--cyclodextrin; channel open probability