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Background and purpose: Vascular ATP-sensitive potassium (KATP) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca2+-dependent phosphatase, inhibits KATP channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA. Experimental approach: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular KATP channel (KIR6.1/SUR2B). The effect of intracellular Ca2+ and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. Key results: Constitutively active calcineurin Aα but not Aβ significantly attenuated KATP currents activated by low intracellular Ca2+, whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal KATP currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca2+ (equivalent to increasing calcineurin activity), almost completely inhibited KATP channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA. Conclusions and implications: Calcineurin Aα regulates KIR6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the KATP channel. British Journal of Pharmacology (2009) 157, 554–564; doi:10.1111/j.1476-5381.2009.00221.x; published online 7 May 2009 This article is commented on by Tammaro, pp. 551–553 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

The prostacyclin (IP) receptor agonists, treprostinil, iloprost and the selexipag metabolite, MRE-269 (ACT-333679) were evaluated in rat distal pulmonary blood vessels. Small pulmonary arteries and veins were pre-contracted with the thromboxane mimetic, U46619 (25 and 100nM, respectively), and relaxation determined with and without IP receptor antagonists, RO1138452 and RO3244794. In arteries, treprostinil was a more potent vasorelaxant than iloprost, while the efficacy of iloprost was greater. In pulmonary arteries, treprostinil-induced relaxation was essentially abolished by both IP antagonists (1μM), while responses to iloprost were partially inhibited. Both treprostinil and iloprost were equipotent, prominently relaxing pulmonary veins with responses being similarly and partially sensitive to IP antagonists. In contrast, RO1138452 failed to inhibit relaxations to MRE-269 in either pulmonary arteries or veins, suggesting no involvement of typical IP receptors. Thus, rat pulmonary tissues cannot be considered appropriate to assess classical IP receptors using the proposed highly selective non-prostanoid agonist MRE-269, contrasting with the IP receptor agonism profile of prostacyclin analogues, iloprost and treprostinil., (Copyright © 2013 Elsevier Inc. All rights reserved.)

ATP-sensitive potassium channels are an octomeric complex of four pore-forming subunits of the Kir 6.0 family and four sulfonylurea receptors. The Kir 6.0 family consists of two known members, Kir 6.1 and Kir 6.2, with distinct functional properties. The tetrameric structure of the pore-forming domain leads to the possibility that mixed heteromultimers may form. In this study, we examine this by using biochemical and electrophysiological techniques after heterologous expression of these subunits in HEK293 cells. After the coexpression of Kir 6.1 and Kir 6.2, Kir 6.1 can be coimmunoprecipitated with isoform-specific Kir 6.2 antisera and vice versa. Coexpression of SUR2B and Kir 6.2 with Kir 6.1 dominant negatives at a 1:1 expression ratio and vice versa led to a potent suppression of current. Kir 6.1, and Kir 6.2 dominant negative mutants were without effect on an inwardly rectifying potassium channel from a different family, Kir 2.1. Single-channel analysis, after coexpression of SUR2B, Kir 6.1, and Kir 6.2, revealed the existence of five distinct populations with differing single-channel current amplitudes. All channel populations were inhibited by glibenclamide. A dimeric Kir 6.1-Kir 6.2 construct expressed with SUR2B had a single-channel conductance intermediate between that of either Kir 6.2 or Kir 6.1 expressed with SUR2B. In conclusion, Kir 6.1 and Kir 6.2 readily coassemble to produce functional channels, and such phenomena may contribute to the diversity of nucleotide-regulated potassium currents seen in native tissues.

ATP-sensitive K(+) (K(ATP)) channels are broadly distributed in the vasculature and regulate arterial tone. These channels are inhibited by intracellular ATP ([ATP](i)) and vasoconstrictor agents and can be activated by vasodilators. It is widely assumed that K(ATP) channels are insensitive to Ca(2+), although regulation has not been examined in the intact cell where cytosolic regulatory processes may be important. Thus we investigated the effects of Ca(2+) on whole-cell K(ATP) current in rat aortic smooth muscle cells recorded in a physiological [ATP](i) and K(+) gradient. Under control recording conditions, cells had a resting potential of approximately -40 mV when bathed in 1.8 mmol/L Ca(2+). The K(ATP) channel inhibitor glibenclamide caused membrane depolarization (9 mV) and inhibited a small, time-independent background current. Reducing [ATP](i) from 3 to 0.1 mmol/L hyperpolarized cells to approximately -60 mV and increased glibenclamide-sensitive current by 2- to 4-fold. Similar effects were observed when Ca(2+) levels were decreased either externally or internally by increasing EGTA from 1 to 10 mmol/L. Dialysis with solutions containing different free [Ca(2+)](i) showed that K(ATP) current was maximally activated at 10 nmol/L [Ca(2+)](i) and almost totally inhibited at 300 nmol/L. Moreover, under control conditions, when rat aortic smooth muscle cells were dialyzed with either cyclosporin A, FK-506, or calcineurin autoinhibitory peptide (structurally unrelated inhibitors of Ca(2+)-dependent protein phosphatase, type 2B), glibenclamide-sensitive currents were large and the resting potential was hyperpolarized by approximately 20 to 25 mV. We report for the first time that K(ATP) channels can be modulated by Ca(2+) at physiological [ATP](i) and conclude that modulation occurs via protein phosphatase type 2B.

The stable prostacyclin analogue, iloprost relaxes a variety of blood vessels and increases cyclic AMP, although the relationship between adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and vasorelaxation remains unclear. We therefore investigated the effect of the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) on iloprost-mediated relaxation and cyclic AMP elevation in endothelium-denuded aortic strips. Iloprost (1-1000 nM) caused a concentration-dependent inhibition of phenylephrine (1-6 microM) contractions, the responses being unaffected by pre-incubation with SQ22536 (100 microM) for 30 min. In other experiments 60 nM iloprost caused a 64% inhibition of phenylephrine contractions concomitant with a 3 fold rise in cyclic AMP. SQ22536 completely abolished the iloprost-induced elevation in cyclic AMP while having no significant effect on relaxation. Our results therefore strongly suggest that cyclic AMP-independent pathways are responsible for the vasorelaxant effects of iloprost in guinea-pig aorta.

Using the patch-clamp recording technique, we observed that endothelin-1 (ET-1; 0.8-16 nM) enhanced a voltage-activated outward current (Iout) and induced periodic oscillations of inward current in smooth muscle cells isolated from small pulmonary arteries (200-400 microns in diameter). Anion substitution experiments revealed that the ET-1-induced inward current was carried by Cl- ions. Application of bosentan (10 microM; and ETA and ETB receptor antagonist) and FR 139317 (1-10 microM; a selective ETA receptor antagonist) prevented initiation of inward currents or enhancement of Iout by ET-1. The ETB receptor agonist tetra-Ala-endothelin-1 (1-20 nM) failed to evoke these responses. Caffeine (10 mM) induced a single transient inward current and prevented any further activation of inward current, or enhancement of Iout, by subsequent application of 16 nM ET-1, suggesting that these currents were mediated by Ca2+ release from internal stores. Rapid intracellular release of Ca2+ by photolysis of nitr-5 activated an inward Cl- current and increased the magnitude of Iout. These results demonstrate the existence of Ca(2+)-activated Cl- and K+ channels in pulmonary arterial smooth muscle. The physiological role of these channels is at present uncertain, although their activation may be involved in the contractile responses of pulmonary arterial smooth muscle to ET-1.

Elevation of intracellular ATP levels by flash photolysis of caged ATP augmented the delayed rectifier K-current (IKDR) in rabbit pulmonary artery myocytes. The percentage augmentation was unaffected when IKDR was inactivated by 50% (holding potential -40 mV), although the magnitude of the ATP-induced current was substantially reduced. Inactivation of 90% IKDR (holding potential -20 mV) virtually abolished the ATP-dependent augmentation. We conclude that modulation of IKDR by ATP does not require conversion of the glibenclamide-sensitive K-current (IK(ATP)).

Large conductance Ca(2+)-activated K+ channels in rabbit pulmonary artery smooth muscle cells are activated by membrane stretch and by arachidonic acid and other fatty acids. Activation by stretch appears to occur by a direct effect of stretch on the channel itself or a closely associated component. In excised inside-out patches stretch activation was seen under conditions which precluded possible mechanisms involving cytosolic factors, release of Ca2+ from intracellular stores, or stretch induced transmembrane flux of Ca2+ or other ions potentially capable of activating the channel. Fatty acids also directly activate this channel. Like stretch activation, fatty acid activation occurs in excised inside-out patches in the absence of cytosolic constituents. Moreover, the channel is activated by fatty acids which, unlike arachidonic acid, are not substrates for the cyclo-oxygenase or lypoxygenase pathways, indicating that oxygenated metabolites do not mediate the response. Thus, four distinct types of stimuli (cytosolic Ca2+, membrane potential, membrane stretch, and fatty acids) can directly affect the activity of this channel.

Single cells from the rabbit pulmonary artery were isolated using a new and convenient procedure. Strips of muscle were incubated overnight in papain at 6 degrees C and dispersed the following morning after warming the tissue for 10 min. This method consistently produced a high yield of relaxed cells, which reversibly responded to vasoconstrictors and remained viable for many hours. The electrophysiological properties of these cells were studied using the patch-clamp technique in the whole-cell configuration. In physiological Ca2+ solution with K(+)-filled pipettes, cells had a high input resistance (approximately 17 G omega) and an average resting potential of -55 mV. In voltage clamp, several components of outward current could be identified. Depolarizing voltage steps revealed a prominent, transient current (Itran), having extremely rapid activation (less than 5 ms) and inactivation (less than 15 ms) kinetics. Itran was followed by a more slowly activating current (IKso) that was sustained over 100 ms. Both currents were essentially abolished by a 4-aminopyridine (4-AP) and sensitive to Ca2+ influx. IKso, but not Itran, was blocked by tetraethylammonium (TEA) and had the properties of a Ca(2+)-activated K+ current. Holding the membrane potential at -40 mV completely inactivated Itran and unmasked a time-independent, background current superimposed on IKso. The background current was also blocked by 4-AP. In addition, when adenosine triphosphate (ATP), but not guanosine triphosphate (GTP), was omitted from the patch-pipette, spontaneous bursts of outward current (SOCs) were superimposed on the voltage-activated currents. However, since SOCs were rarely observed when ATP and GTP were present together, they are unlikely to be active under physiological conditions. Thus at least four types of outward current can be distinguished in isolated rabbit pulmonary artery cells. These include a novel transient current which could be activated from the resting potential. It activates much more rapidly than outward currents previously reported in vascular muscle, and would rapidly oppose action potential firing. This current could therefore be responsible for the inability of large elastic arteries to fire action potentials.

The effects of the beta-adrenergic agent isoproterenol on membrane currents were studied in freshly dissociated gastric smooth muscle cells of Bufo marinus. Voltage-clamp experiments were carried out with patch pipettes in the tight-seal, whole-cell recording mode or with conventional microelectrodes. Isoproterenol induced a current identified as M current by the following criteria: the induced current is outward and carried by K+ ions, is suppressed by muscarine or acetylcholine, remains steadily activated, turns off with hyperpolarization, and exhibits slow relaxations in response to voltage jumps. In contrast to endogenous M current, isoproterenol-induced M current usually exhibited slower relaxations on hyperpolarizing voltage commands and displayed a steady-state conductance/voltage relationship that was shifted in the negative direction along the voltage axis. M current was also induced by either forskolin or phosphodiesterase-resistant cAMP analogs. In all cases, muscarinic agonists suppressed the M current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. beta-Adrenergic agents may act to increase the number of M channels available to be opened and also modify their kinetics.

Electrophysiological recordings from freshly-dissociated smooth muscle cells from toad stomach revealed that substance P enhances one of two types of Ca2+ currents. That is, substance P enhances the slowly inactivating, high-threshold current but not the fast inactivating, low-threshold current. Acetylcholine has the same effect, but the acetylcholine action is blocked by atropine whereas the substance P action is not, indicating that the two agents act at different receptor sites. Thus, substance P, like acetylcholine, has a dual excitatory action on the smooth muscle cells employed in these studies, enhancing a specific type of Ca2+ current, as demonstrated here, and suppressing a voltage-sensitive K+ conductance, as previously described [Sims, S.M., Walsh, J.V., Jr. & Singer, J.J. (1986) Am. J. Physiol. 251, C580-C587].

Electrophysiological recordings from freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus revealed two types of Ca2+ currents. One has a low threshold of activation and inactivates rapidly; the other has a high threshold of activation and inactivates more slowly. Acetylcholine (ACh) increased the high-threshold current but not the low-threshold current. The synthetic diacylglycerol analog sn-1,2-dioctanoylglycerol, an activator of protein kinase C (PKC), mimicked these effects of ACh on Ca2+ currents. However, another diacylglycerol analog, 1,2-dioctanoyl-3-thioglycerol, which has a closely related structure but does not activate PKC, failed to increase the Ca2+ current. The same was true of 1,2-dioctanoyl-3-chloropropanediol, an analog that even at high concentrations only minimally activates PKC. These results suggest that diacylglycerol may be the second messenger mediating the effects of ACh on one type of voltage-activated Ca2+ channel, possibly by activating PKC.

In helical strips cut from the small mesenteric artery of guinea-pig (GPSMA) (0.3-0.6 mm o.d.) relaxations induced by substance P were more susceptible to damage of the endothelium by rubbing than were relaxations evoked by carbachol. Relaxations induced by 2-nicotin-amidoethyl nitrate (SG75) were unaffected by this procedure. Relaxations evoked by the calcium ionophore A23187 persisted when those to substance P had been abolished by rubbing the endothelium in GPSMA, rabbit mesenteric and rabbit ear arteries. In guinea-pig pulmonary artery and aorta relaxations to A23187 were lost after this treatment. Carbachol and SG75 were more effective in inhibiting phasic than tonic tension induced by noradrenaline in GPSMA, but substance P was more effective against tonic tension. In the GPSMA, carbachol and substance P inhibited tension produced by noradrenaline to similar extents. However, carbachol was less, and substance P much less effective in inhibiting tension evoked by high-potassium solution than by noradrenaline. Susceptibility of relaxations to blockade by haemoglobin in GPSMA was: substance P greater than carbachol greater than ATP greater than SG75. The membrane potential of smooth muscle cells in the media of the GPSMA was recorded by microelectrode. Carbachol, but not substance P, hyperpolarized the cells both in the presence and absence of noradrenaline at concentrations which relaxed the muscle. These results suggest a heterogeneity in the mechanisms of endothelial-dependent relaxations induced by various vascular relaxants.