Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.     

Purinergic P2Y6 receptors induce Ca2+ and CFTR dependent Cl- secretion in mouse trachea.

In airways Cl- secretion is activated and Na+ absorption is inhibited when P2Y2 receptors are stimulated by ATP or UTP. Both nucleotides are subject to degradation to ADP and UDP by ecto-nucleotidases. Here we show that these metabolites change electrolyte transport by stimulation of P2Y6 receptors in mouse trachea. Immunohistochemistry confirmed luminal and basolateral expression of P2Y6 receptors. In Ussing chamber experiments luminal ADP, UDP or the P2Y6 receptor agonist INS48823 induced both transient and persistent increase in short circuit currents (ISC). Activation of ISC was inhibited by the P2Y6 receptor blocker PPADS. The transient response was inhibited by DIDS, whereas the persistent ISC was inhibited by glibenclamide and by the protein kinase A (PKA) blocker H-89. Moreover, sustained activation of ISC by luminal UDP was inhibited by blocking basolateral K+ channels with 293B. Possible effects of diphosphates on P2Y1 or adenosine receptors were excluded by the inhibitors MRS2179 and 8-SPT, respectively. Inhibition of amiloride sensitive Na+ absorption was only seen after blocking basolateral K+ channels with 293B. In contrast, Cl- secretion activated by basolateral ADP or UDP was only transient and was blocked by the sk4 K+ channel blocker clotrimazole. In summary, activation of luminal P2Y6 receptors in the airways shifts electrolyte transport towards secretion by increasing intracellular Ca+ and activation of PKA.     

Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current (Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.     

Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon

Studies from our laboratory have demonstrated rapid ( < 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC activity and a PKC-dependent Ca(2+) entry through L-type Ca(2+) channels specifically by mineralocorticoids in distal colon. Aldosterone directly stimulates the activity of the PKC alpha isoform (but not PKC delta, PKC epsilon and PKC zeta) in a cell-free assay system containing only purified commercially available enzyme, appropriate substrate peptide, co-factors and lipid vesicles. The primary ion transport target of the non-genomic signal transduction cascade elicited by aldosterone in epithelia is the Na(+)-H(+) exchanger. In isolated colonic crypts, aldosterone produced a PKC alpha sensitive intracellular alkalinisation within 1 min of hormone addition. Intracellular alkalinisation upregulates an ATP-dependent K(+) channel, which is involved in K(+) recycling to maintain the electrical driving force for Na(+) absorption, while inhibiting a Ca(2+) -dependent K(+) channel, which generates the charge balance for Cl(-) secretion. The non-genomic response to aldosterone in distal colon appears to enhance the capacity for absorption while down-regulating the potential for secretion. We have also demonstrated rapid (< 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC alpha activity, and a PKC delta- and PKA-dependent Ca(2+) entry through di-hydropyridine-blockable Ca(2+) channels specifically by 17beta-estradiol in distal colon. These rapid effects are female gender specific and are insensitive to inhibitors of the classical estrogen receptor (ER). 17 beta-Estradiol directly stimulated the activity of both PKC delta and PKC alpha (but not PKC epsilon or PKC zeta) in a cell-free assay system. E2 rapidly inhibited basolateral K(Ca) channel activity which would be expected to result in an acute inhibition of Cl(-) secretion. Physiological concentrations of E2 (0.1-10 nM) reduced both basal and secretagogue-induced Cl(-) secretion. This anti-secretory effect of E2 is sensitive to PKC inhibition, intracellular Ca(2+) chelation, and is female gender specific and insensitive to inhibitors of the classical ER. These observations link rapid non-genomic activation of second messengers with a rapid gender-specific physiological effect in the whole tissue. Aldosterone and E2 differ in their protein kinase signal transduction and both hormones stimulate specific PKC isoforms indicating both common and divergent signalling systems for salt-retaining steroid hormones. The physiological function of non-genomic effects of aldosterone and estradiol is to shift the balance from net secretion to net absorption in a pluripotential epithelium.     

Role of voltage-gated calcium channels in potassium-stimulated aldosterone secretion from rat adrenal zona glomerulosa cells

The mineralocorticoid aldosterone plays an important role in the regulation of plasma electrolyte homeostasis. Exposure of acutely isolated rat adrenal zona glomerulosa cells to elevated K(+) activates voltage-gated calcium channels and initiates a calcium-dependent increase in aldosterone synthesis. We developed a novel 96-well format aldosterone secretion assay to rapidly evaluate the effect of known T- and L-type calcium channel antagonists on K(+)-stimulated aldosterone secretion and better define the role of voltage-gated calcium channels in this process. Reported T-type antagonists, mibefradil and Ni(2+), and selected L-type antagonist dihydropyridines, inhibited K(+)-stimulated aldosterone synthesis. Dihydropyridine-mediated inhibition occurred at concentrations which had no effect on rat alpha1H T-type Ca(2+) currents. In contrast, below 10 microM, the L-type antagonists verapamil and diltiazem showed only minimal inhibitory effects. To examine the selectivity of the calcium channel antagonist-mediated inhibition, we established an aldosterone secretion assay in which 8Br-cAMP stimulates aldosterone secretion independent of extracellular calcium. Mibefradil remained inhibitory in this assay, while the dihydropyridines had only limited effects. Taken together, these data demonstrate a role for the L-type calcium channel in K(+)-stimulated aldosterone secretion. Further, they confirm the need for selective T-type calcium channel antagonists to better address the role of T-type channels in K(+)-stimulated aldosterone secretion.     

Role of protein kinase C in aldosterone-induced non-genomic inhibition of basolateral potassium channels in human colonic crypts

Aldosterone produces rapid, non-genomic, inhibition of basolateral intermediate conductance K(+) (IK(Ca)) channels in human colonic crypt cells but the intracellular second messengers involved are unclear. We therefore evaluated the role of protein kinase C (PKC) in aldosterone's non-genomic inhibitory effect on basolateral IK(Ca) channels in crypt cells from normal human sigmoid colon. Patch clamp studies revealed that in cell-attached patches, IK(Ca) channel activity decreased progressively to 38+/-8% (P<0.001) of the basal value 10 min after the addition of 1 nmol/L aldosterone, and decreased further to 23+/-6% (P<0.02) of the basal value 5 min after increasing the aldosterone concentration to 10 nmol/L. Pre-incubation of crypts with 1 micromol/L chelerythrine chloride or 1 micromol/L G? 6976 (PKC inhibitors) prevented the inhibitory effect of aldosterone. Conversely, channel activity decreased to 60+/-9% (P<0.02) of the basal value 10 min after the addition of 500 nmol/L PMA (a PKC activator), whereas 4alpha-PMA (an inactive ester) had no effect. When aldosterone (10 nmol/L) and PMA were added together, IK(Ca) channel activity was inhibited to the same extent as with aldosterone alone. These results indicate that aldosterone's non-genomic inhibitory effect on the macroscopic basolateral K(+) conductance in human colonic crypts reflects PKC-mediated inhibition of IK(Ca) channels.     

Basolateral store-operated Ca(2+)-entry in polarized human bronchial and colonic epithelial cells

Bronchial epithelial cells respond to extracellular nucleotides from the luminal and basolateral side activating Cl- secretion via [Ca2+]i increase. In this study we investigated the differences of apically (ap) and basolaterally (bl) stimulated [Ca2+]i signals in polarized human bronchial epithelial cells (16HBE14o-). Specifically we investigated the localization of 'capacitative Ca2+ entry' (CCE). 16HBE14o- cells grown on permeable filters were mounted into an Ussing chamber built for the simultaneous measurement of Fura-2 fluorescence and electrical properties. Application of ATP from both sides induced a rapid [Ca2+]i increase and subsequent sustained [Ca2+]i plateau due to transmembraneous Ca(2+)-influx. The use of different nucleotides revealed the following rank order or potency which was very similar for addition from the apical or basolateral side: UTP (EC50 ap: 4 microM, bl: 5 microM) > ATP (EC50 ap: 4 microM, bl: 10 microM) > ADP (n = 4-7 from both sides). 2-MeS-ATP, AMP, adenosine and beta gamma-methylene ATP were ineffective (n = 3 from both sides). The ATP- (ap and bl) induced Ca2+ influx was only abolished by removal of basolateral Ca2+. This was also true for receptor-independent activation of Ca(2+)-influx by intracellular Ca(2+)-store depletion with 2,5 Di-(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 microM). Also in polarized T84 cells the basolateral carbachol and BHQ activated Ca2+ plateau was exclusively sensitive to removal of basolateral Ca2+. We propose that in all polarized epithelial cells the CCE entry pathway is located in the basolateral membrane. We furthermore suggest that Ca2+[i elevating agonists acting from the apical side of the epithelium lead to the opening of a basolateral CCE pathway.     

Desensitization of pituitary gonadotropin secretion by agonist-induced inactivation of voltage-sensitive calcium channels

Gonadotropin-releasing hormone (GnRH) stimulates calcium mobilization and influx in pituitary gonadotrophs, and agonist-induced calcium entry through voltage-sensitive channels (VSCC) is required for the maintenance of gonadotropin secretion. However, prolonged or frequent exposure to GnRH attenuates the extracellular Ca2+-dependent cytosolic Ca2+ signal and diminishes hormone secretion. Measurements of membrane Ca2+ currents revealed significant impairment of VSCC activity in gonadotrophs during desensitization by GnRH. VSSC were also inactivated in a calcium-dependent manner during exposure to high K+. Prolonged inactivation of such Ca2+ channels by high K+ reduced the calcium and secretory responses to GnRH and vice versa. The calcium-dependent inactivation of VSCC during GnRH action appears to be a primary factor in the onset of desensitization in pituitary gonadotrophs. This mechanism could also account for the development of agonist-induced refractoriness in other calcium-regulated target cells.     

Potassium channel activity recorded from the apical membrane of freshly isolated epithelial cells in rat caudal epididymis.

K+ channels were recorded in excised, inside-out patches from the apical membrane of the freshly isolated tubule of the caudal portion of the rat epididymis. With asymmetric K+ concentrations in bath and pipette (140 mM K+in/6 mM K+out), the channels had a slope conductance of 54.2 pS at 0 mV. The relative permeability of K+ over Na+ was about 171 to 1. The channels were activated by intracellular Ca2+ and by membrane depolarization. These channels belong to a class defined as "intermediate-conductance Ca2+-activated K+ channel. " External tetraethylammonium ions (TEA+) caused a flickery block of the channel with reduction in single-channel current amplitude measured at a range of holding membrane potentials (-40 to 60 mV). Activity of the K+ channels was inhibited by intracellular ATP (KD =1.188 mM). The channel activity was detected only occasionally in patches from the apical membrane (about 1 in 17 patches containing active channels). The presence of the intermediate-conductance Ca2+-activated K+ channels indicates that they could provide a route for K+ secretion in a Ca2+-dependent process responsible for a high luminal K+ concentration found in the epididymal duct of the rat.     

Control of ion transport in mouse proximal and distal colon by prolactin.

The lactogenic hormone prolactin (PRL) has been known to affect Ca(2+) and electrolyte transport in the intestinal epithelium. In the present study we analyzed ion transport in mouse proximal and distal colon, and acute changes induced by PRL. In the proximal colon, carbachol activated a Ca(2+) dependent Cl(-) secretion that was sensitive to DIDS and NFA. In the distal colon, both ATP and carbachol activated K(+) secretion. Ca(2+) -activated KCl transport in proximal and distal colon was inhibited by PRL (200 ng/ml), while amiloride sensitive Na(+) absorption and cAMP induced Cl(-) secretion remained unaffected. Luminal large conductance Ca(2+) -activated K(+) (BK) channels were largely responsible for Ca(2+) -activated K(+) secretion in the distal colon, and basolateral BK channels supported Ca(2+) -activated Cl(-) secretion in the proximal colon. Ca(2+) chelating by BAPTA-AM attenuated effects of carbachol and abolished effects of PRL. Both inhibition of PI3 kinase with wortmannin and blockage of MAP kinases with SB 203580 or U 0126, interfered with the acute inhibitory effect of PRL on ion transport, while blocking of Jak/Stat kinases with AG 490 was without effects. PRL attenuated the increase in intracellular Ca(2+) that was caused by stimulation of isolated colonic crypts with carbachol. Thus PRL inhibits Ca(2+) dependent Cl(-) and K(+) secretion by interfering with intracellular Ca(2+) signaling and probably by activating PI3 kinase and MAP kinase pathways.     

Role of GPR40 in fatty acid action on the beta cell line INS-1E

GPR40 is a G protein-coupled receptor expressed preferentially in beta cells, that has been implicated in mediating free fatty acid-stimulated insulin release. GPR40 RNAi impaired the ability of palmitic acid (PA) to increase both insulin secretion and intracellular calcium ([Ca2+]i). The PA-dependent [Ca2+]i increase was attenuated by inhibitors of Galphaq, PLC, and SERCA. Thus GPR40 activates the Galphaq pathway, leading to release of Ca2+ from the ER. Yet the GPR40-dependent [Ca2+]i rise was dependent on extracellular Ca2+ and elevated glucose, and was blocked by inhibition of L-type calcium channels (LTCC) or opening of the K(ATP) channel; this suggests that GPR40 promotes Ca2+ influx through up-regulation of LTCC pre-activated by glucose and membrane depolarization. Taken together, the data indicate that GPR40 mediates the increase in [Ca2+]i and insulin secretion through the Galphaq-PLC pathway, resulting in release of Ca2+ from the ER and leading to up-regulation of Ca2+ influx via LTCC.     

Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion

In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence different types of K(+) channels mediate basolateral K(+) exit during transepithelial Na(+) and Cl(-) transport.     

Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.

The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.     

Mechanism of inhibitory action of TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] on aldosterone secretion in adrenal glomerulosa cells.

The mechanism of 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) action was evaluated in isolated adrenal glomerulosa cells. TMB-8 inhibits both angiotensin II- and K+-stimulated aldosterone secretion in a dose-dependent manner. The ID50 for angiotensin II- and K+-stimulated aldosterone secretion is 46 and 28 microM, respectively. In spite of the fact that 100 microM-TMB-8 inhibits angiotensin II-stimulated aldosterone secretion almost completely, TMB-8 (100 microM) does not inhibit angiotensin II-induced 45Ca2+ efflux from prelabelled cells nor does it affect inositol 1,4,5-trisphosphate-induced calcium release from non-mitochondrial pool(s) in saponin-permeabilized cells. TMB-8 has no inhibitory effect on A23187-induced aldosterone secretion, but 12-O-tetradecanoylphorbol 13-acetate-induced aldosterone secretion is completely abolished. TMB-8 effectively inhibits both angiotensin II- and K+-induced increases in calcium influx but has no effect on A23187-induced calcium influx. TMB-8 inhibits the activity of protein kinase C dose-dependently. These results indicate that TMB-8 inhibits aldosterone secretion without inhibiting mobilization of calcium from an intracellular pool. The inhibitory effect of TMB-8 is due largely to an inhibition of plasma membrane calcium influx, but this drug also inhibits the activity of protein kinase C directly.     

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 microM-1 mM and was induced at different values of intracellular pH and Ca2+. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg2+ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca2+ and their sensitivity to intracellular cAMP are consistent with a role in the Ca2+ oscillations associated with cell growth.     

ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors

This study describes a novel mechanism of regulation of the high-affinity Na(+)-dependent adenosine transporter (CNT2) via the activation of A(1) adenosine receptors (A(1)R). This regulation is mediated by the activation of ATP-sensitive K(+) (K(ATP)) channels. The high-affinity Na(+)-dependent adenosine transporter CNT2 and A(1)R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (-)-N(6)-(2-phenylisopropyl)adenosine is fully inhibited by K(ATP) channel blockers and mimicked by a K(ATP) channel opener. A(1)R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A(1)R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A(1)R, CNT2, and K(ATP) channels. These results also suggest that the activation of K(ATP) channels under metabolic stress can be mediated by the activation of A(1)R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for K(ATP) channels.     

Extracellular ATP activates receptor-operated cation channels in mouse lacrimal acinar cells to promote calcium influx in the absence of phosphoinositide metabolism.

In exocrine acinar cells a variety of neurotransmitters (e.g. acetylcholine) stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis elevating intracellular calcium to activate calcium-dependent membrane currents (outward K+ and inward Cl-). This study shows that in lacrimal acinar cells extracellular application of ATP is also associated with outward and inward current responses; these, however, are not the result of phosphoinositide metabolism. ATP directly activates receptor-operated cation channels which permit influx of Na+ and Ca+ (the inward current). The elevation in [Ca2+]i which results is sufficient to activate the outward K+ current. ATP thus promotes Ca+ influx in the absence of phosphoinositide metabolism.     

Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules

beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.