Differential actions of the prostacyclin analogues treprostinil and iloprost and the selexipag metabolite, MRE-269 (ACT-333679) in rat small pulmonary arteries and veins.

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.

Ca2+/calcineurin regulation of cloned vascular K ATP channels: crosstalk with the protein kinase A pathway.

BACKGROUND AND PURPOSE: Vascular ATP-sensitive potassium (K(ATP)) 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 Ca(2+)-dependent phosphatase, inhibits K(ATP) 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 K(ATP) channel (K(IR)6.1/SUR2B). The effect of intracellular Ca(2+) and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. KEY RESULTS: Constitutively active calcineurin A alpha but not A beta significantly attenuated K(ATP) currents activated by low intracellular Ca(2+), whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal K(ATP) currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca(2+) (equivalent to increasing calcineurin activity), almost completely inhibited K(ATP) 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 A alpha regulates K(IR)6.1/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 K(ATP) channel.

Temporal variation in endotoxin-induced vascular hyporeactivity in a rat mesenteric artery organ culture model.

Endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is well described in rodent aorta, but has not been investigated in smaller vessels in vitro. Segments of rat superior mesenteric artery were incubated in culture medium with or without foetal bovine serum (10%) for 6, 20 or 46 h in the presence or absence of bacterial lipopolysaccharide (LPS; 1 - 100 microg ml(-1)). Contractions to PE were measured with or without nitric oxide synthase (NOS) inhibitors: L-NAME (300 microM), aminoguanidine (AMG; 400 microM) 1400W (10 microM) and GW273629 (10 microM); the guanylyl cyclase inhibitor, ODQ (3 microM); the COX-2 inhibitor, NS-398 (10 microM). Contractile responses to the thromboxane A2 mimetic, U46619 were also assessed. In the presence of serum, LPS induced hyporeactivity at all time points. In its absence, hyporeactivity only occurred at 6 and 20 h. L-NAME and AMG fully reversed hyporeactivity at 6 h, whereas they were only partially effective at 20 h and not at all at 46 h. In contrast partial reversal of peak contraction was observed with 1400W (62% at 46 h), GW273629 (57% at 46 h) and ODQ (75% at 46 h). COX-2 inhibition produced no reversal. In contrast to PE, contractions to U46619 were substantially less affected by LPS. We describe a well-characterized reproducible model of LPS-induced hyporeactivity, which is largely mediated by the NO-cyclic GMP-dependent pathway. Importantly, long-term (2-day) production of NO via iNOS is demonstrated. Moreover, conventional doses of L-NAME and AMG became increasingly ineffective over time. Thus, the choice of inhibitor merits careful consideration in long-term models.

A mechanism for ATP-sensitive potassium channel diversity: Functional coassembly of two pore-forming subunits.

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.

Calcium modulation of vascular smooth muscle ATP-sensitive K(+) channels: role of protein phosphatase-2B.

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.

Effects of the adenylyl cyclase inhibitor SQ22536 on iloprost-induced vasorelaxation and cyclic AMP elevation in isolated guinea-pig aorta.

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.

Evidence that Ca2+-activated K+ channels play a major role in mediating the vascular effects of iloprost and cicaprost.

The role of K+ channels in mediating vasorelaxation induced by two prostacyclin analogues was investigated in guinea-pig aorta. Iloprost caused substantial relaxation of tissues contracted with phenylephrine or 25 mM K+ but not 60 mM K+. In endothelial-denuded tissues, maximal relaxations to iloprost, cicaprost or isoprenaline were inhibited by approximately 40-50% with tetraethylammonium or iberiotoxin, both blockers of large conductance Ca2+-activated K+ (BKCa) channels. In contrast, the response to forskolin, an activator of adenylate cyclase was marginally inhibited by tetraethylammonium. The K(ATP) channel blocker, glibenclamide significantly augmented the response to iloprost but not cicaprost. These effects were largely inhibited by the EP1 receptor antagonist, 8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid 2-[1-oxo-3(4-pyridinyl)propyl]hydrazide, monohydrochloride (SC-51089) and partially by indomethacin, suggesting that iloprost relaxation is counterbalanced by activation of EP1 receptors, in part through a constrictor prostaglandin. We conclude that BKCa channels play an important role in mediating the effects of iloprost and cicaprost and raises the possibility that cyclic AMP-independent pathways might be involved.

Potassium channels in the vasculature.

Many important cellular functions are regulated by vascular potassium channels, including the resting membrane potential. Recent evidence suggests that the function of these channels is altered in pathophysiological disorders of the cardiovascular system. Using molecular cloning techniques, considerable effort has been made over the past 5 years to elucidate the structure of various types of potassium channels. Several different potassium channel clones have been identified from neuronal and cardiac tissues, although only a few have so far been identified in smooth muscle.

Ca(2+)-activated Cl- currents in pulmonary arterial myocytes.

Currents from smooth muscle cells isolated from the pulmonary arterial tree of the rat were recorded under voltage clamp using the whole cell configuration of the patch-clamp technique. Rapid increases in cytosolic free calcium evoked by flash photolysis of Nitr-5 activated a current that, following ion substitution and pharmacological experiments, proved to be carried by Cl-. This current [ICl(Ca)] was evoked independently of photolytic by-products and, although smaller, was still activated in the absence of pipette ATP. Experiments revealed that ICl(Ca) was evoked in 80% in the cells isolated from the main pulmonary artery but only in 43% of the cells isolated from small vessels (200-400 microns ID). Application of caffeine also resulted in activation of ICl(ca), although the response current magnitude was larger in the main pulmonary artery. Photolysis of Nitr-5 still activated ICl(ca) in the presence of caffeine, suggesting that Ca2-release is not a prerequisite for activation of ICl(ca). These results represents in the first electrophysiological recordings of Cl- currents from small pulmonary arterial vessels and indicate that their Ca2+ regulation and/or distribution may be different throughout the pulmonary circulation.

Regulation of glibenclamide-sensitive K+ current by nucleotide phosphates in isolated rabbit pulmonary myocytes.

OBJECTIVE: Although the existence of ATP-sensitive K+ (KATP) channels in vascular muscle is widely accepted, there appears to be little consensus as to what the primary regulator of these channels is under physiological or pathophysiological conditions. Recent evidence has suggested that nucleotide diphosphates (NDPs) may play a more important role than ATP. However, since the properties of vascular KATP channels are quite diverse, and the effects of these nucleotides are poorly understood, the aim of this study was to test the hypothesis that intracellular ATP can regulate whole-cell KATP current (IK,ATP) in the absence of NDPs. METHODS: Single cells were isolated from rabbit main pulmonary artery by enzymatic treatment with papain. Whole-cell patch clamp experiments were performed in cells dialysed with different nucleotides. The effect of the KATP channel activator, levcromakalim (10 microM), was investigated at a holding potential of -60 mV. The contribution of IK,ATP to the holding current was defined as the current which was blocked by glibenclamide following washout of levcromakalim. RESULTS: Lowering the intracellular ATP concentration ([ATP]i) from 1 to 0.1 mM, in the presence or absence of GTP, enhanced the levcromakalim-induced current (Ilev) by approximately 2.5 fold and increased a glibenclamide-sensitive background K+ current (Iglib). However, Iglib was larger with GTP and the total glibenclamide-sensitive current (Ilev+Iglib) increased with time. Significant activation of Iglib failed to occur when the pipette contained no nucleotides and the responses to levcromakalim were generally much smaller than seen with ATP. GDP (0.5 mM), in the absence of pipette ATP, activated a large background K+ current which had similar properties to Ilev. Consistent with this was the observation that Ilev became substantially reduced in the presence of GDP, presumably because a significant amount of IK,ATP was already activated. CONCLUSIONS: The response to levcromakalim in isolated cells from pulmonary artery was, as expected for an agent activating KATP channels, modulated by changes in the pipette [ATP]. This effect was not dependent on the presence of other pipette nucleotides, although the possibility cannot be excluded that metabolites from the cellular breakdown of ATP are essential for normal channel regulation. GDP could also activate IK,ATP under conditions where the channel is probably in a low phosphorylation state. The time-dependent effects of GTP require further work to determine the precise mechanism, but may suggest that GTP and/or G-proteins are involved in the regulation of KATP channels.

Ca(2+)-activated Cl- and K+ channels and their modulation by endothelin-1 in rat pulmonary arterial smooth muscle cells.

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.

Properties of the ATP-sensitive K+ current activated by levcromakalim in isolated pulmonary arterial myocytes.

Tension and patch clamp recording techniques were used to investigate the relaxation of rabbit pulmonary artery and the properties of the K+ current activated by levcromakalim in isolated myocytes. Under whole-cell voltage clamp, holding at -60 mV in symmetrical 139 mM K+, levcromakalim (10 microM) induced a noisy inward current of -116 +/- 19 pA (n = 13) which developed over 1 to 2 min. This current could be blocked by either glibenclamide (10 microM) or phencyclidine (5-50 microM) and was unaffected when extracellular Ca2+ was removed. Both these drugs inhibited the levcromakalim-induced relaxation of muscle strips precontracted with 20 mM [K+]o. Application of voltage ramps in symmetrical 139 mM K+ confirmed that the levcromakalim-induced current was carried by K+ ions and was weakly voltage dependent over the potential range from -100 to +40 mV. The unitary current amplitude and density of the channels underlying the levcromakalim-activated whole-cell K+ current was estimated from the noise in the current record. We estimate that levcromakalim caused activation of around 300 channels per cell, with a single channel current of 1.1 pA, corresponding to a slope conductance of about 19 pS. Furthermore, cells dialyzed with an ATP-free pipette solution developed a large noisy inward current at -60 mV, which could subsequently be blocked by flash photolysis of caged ATP. Analysis of the noise associated with this current indicated that the single channel amplitude underlying the ATP-blocked current was 1.4 pA, a value similar to that estimated for the levcromakalim-induced current.(ABSTRACT TRUNCATED AT 250 WORDS)

Augmentation by intracellular ATP of the delayed rectifier current independently of the glibenclamide-sensitive K-current in rabbit arterial myocytes.

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)).

ATP-sensitive K+ channels mediate vasodilation produced by lemakalim in rabbit pulmonary artery.

Tension recording and the patch-clamp technique were used to determine the mechanism underlying vasodilation produced by lemakalim in the rabbit pulmonary artery. Lemakalim produced relaxation of precontracted muscle strips that was inhibited by glibenclamide and tetrapentylammonium ions but not by 2 mM tetraethylammonium (TEA) ions. In single cells dialyzed with 1 mM ATP, lemakalim (10 microM) hyperpolarized cells by approximately 13 mV and activated a time-independent K+ current, averaging only 6.5 pA at -50 mV. Glibenclamide reversed both of these membrane effects of lemakalim but not the lemakalim-induced block of an outward current seen above -20 mV. ATP depletion hyperpolarized cells and selectively unmasked a background K+ current, which was sensitive to glibenclamide but not to TEA, with properties similar to the current activated by lemakalim during membrane hyperpolarization. Furthermore, when intracellular ATP concentrations were varied, a clear correlation was revealed between ATP levels and the magnitude of the depolarization or hyperpolarization seen with either glibenclamide or lemakalim, respectively. These results provide direct evidence that the background current is carried by ATP-sensitive K+ channels rather than by large-conductance Ca(2+)-activated K+ channels and that it underlies the hyperpolarization and relaxation to lemakalim.

Role for diacylglycerol in mediating the actions of ACh on M-current in gastric smooth muscle cells.

The role of the second messenger diacylglycerol (DAG) in mediating muscarinic suppression of M-current, a type of a voltage-gated K+ current that is suppressed by acetylcholine (ACh), was examined in freshly isolated smooth muscle cells from toad stomach. Currents were recorded using a single electrode voltage clamp employing conventional microelectrodes. Extracellular application of 1,2-dioctanoyl-sn-glycerol (DiC8), a synthetic DAG that is a potent activator of protein kinase C (PKC), reversibly suppressed M-current. Current relaxations, representing the voltage-dependent closure of K+ channels underlying M-current, were also decreased by DiC8, although suppression was not always as complete as it was with ACh. In contrast, another DAG analogue, 1,2-dioctanoyl-3-thioglycerol, which has a structure closely related to DiC8 but does not activate PKC, failed to inhibit M-current. Furthermore, M-current induced by the beta-agonist isoproterenol, by a mechanism apparently mediated by adenosine 3',5'-cyclic monophosphate (S. M. Sims, L. H. Clapp, J. V. Walsh, Jr., and J. J. Singer. Pflugers Arch. 417: 291, 1990), was also suppressed by DiC8. Both ACh and DiC8 were found to suppress endogenous and isoproterenol-induced M-current without altering the time course of M-current deactivation, suggesting that these agents act by decreasing the number of channels available to be opened. These results provide evidence that muscarinic regulation of M-current is mediated by DAG.

ATP-sensitive K+ channels regulate resting potential of pulmonary arterial smooth muscle cells.

ATP-sensitive K+ (KATP) channels have been proposed to be the target for hyperpolarizing vasodilators. However, the existence of a whole cell KATP current that can regulate membrane potential has not been demonstrated in vascular muscle. Using the patch-clamp technique, we have examined the effects of varying intracellular ATP on membrane potential and currents in isolated rabbit pulmonary arterial smooth muscle cells. With 1 mM ATP in the pipette, cells had a mean resting potential of -55 mV. When ATP was omitted, the resting potential became significantly more hyperpolarized (-70 mV) and the depolarizing response to the KATP-channel blocker, glibenclamide, was potentiated. In contrast, the hyperpolarizing effect of lemakalim was reduced. These hyperpolarized resting potentials were associated with increased activity of a basal, glibenclamide-sensitive time-independent K+ current. Furthermore, flash photolysis of ATP, 3-O-[1(4,5-dimethoxy-2-nitrophenyl)ethyl] ester, disodium salt ("caged ATP") in ATP-depleted cells caused rapid depolarization (less than 1 s) and block of the background K+ current. Our results are consistent with the idea that intracellular ATP can directly modulate the resting potential by inhibition of K+ channels. We propose that this ATP-sensitive K+ current plays an important role in the maintenance of the resting potential in arterial muscle.

Both membrane stretch and fatty acids directly activate large conductance Ca(2+)-activated K+ channels in vascular smooth muscle cells.

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.

Outward currents in rabbit pulmonary artery cells dissociated with a new technique.

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.

Modulation of calcium movements by nitroprusside in isolated vascular smooth muscle cells.

Using the patch-clamp technique, we have characterised the inward current from enzymatically dispersed rabbit pulmonary arterial cells, and investigated the effects of the vasodilator, nitroprusside (NP), on these and other membrane currents. With Cs(+)-filled pipettes, inward currents were recorded during brief depolarizing voltage steps in both physiological Ca2+ and 10 mM Ba2+. The threshold for current activation was positive to -40 mV and the current peaked at 0 mV for Ca2+ and +10 mV for Ba2+. During the first few minutes of recording, inward currents increased or "ran-up". This could not be attributed to blockade of outward current or the inclusion of adenosine triphosphate (ATP) in the patch pipette. Experiments revealed that all the inward current was carried through a single type of voltage-activated Ca2+ channel, namely the high-threshold, dihydropyridine-sensitive channel. It was unaffected by tetrodotoxin but was abolished at all potentials by low concentrations of Cd2+ (100 microM) or nifedipine (1-2 microM). NP (1 microM) suppressed peak inward Ba2+ current at +10 mV by approximately 45%. Higher concentrations (50 microM) did not produce further blockade of the current. This decrease was associated with increased inactivation of the current, and both effects required the presence of ATP in the patch pipette. In physiological Ca2+, using K(+)-filled pipettes, NP was found to induce spontaneous bursts of outward currents, which are probably activated by the release of Ca2+ from Ca(2+)-overloaded stores. These results are consistent with NP lowering cytosolic Ca2+, and hence causing vasodilation, by inhibiting Ca2+ influx through voltage-gated Ca2+ channels and by promoting Ca2+ uptake into the sarcoplasmic reticulum.

Dual regulation of M current in gastric smooth muscle cells: beta-adrenergic-muscarinic antagonism.

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.