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Cell swelling is associated with the activation of an increase in the osmosensitive taurine release (OTR) rate, which serves to decrease cell volume as part of a process known as regulatory volume decrease. OTR, which is sensitive to many pharmacological agents including anion channel blockers and signalling pathway modulators, has also been suggested to play a role in cell cycle progression. At non-cytotoxic concentrations, the anion channel blocker NPPB (25 microM), the extra-cellular signal-regulated kinase inhibitor PD98059 (50 microM), and the c-Jun NH2-terminal kinase inhibitor SP 600125 (5 microM) each decreased the OTR rate by > or =50%, decreased cell proliferation, and increased G0/G1 cell cycle arrest.

A novel anionic background conductance (I(AB)) in cardiac ventricular myocytes has recently been identified but at present there is comparatively little information on its pharmacological modulation. This study investigated the effects of on I(AB) of four pyrethroid agents tefluthrin (a selective activator of this current), tetramethrin, fenpropathrin and alpha-cypermethrin in addition to other well known chloride channel modulators (chlorotoxin, gadolinium and picrotoxin). Guinea-pig ventricular myocytes were isolated using an enzymatic and mechanical dispersion procedure and all electrophysiological measurements were made using the whole-cell patch-clamp technique. In contrast to other anion conductances (stretch- or volume-regulated chloride current (I(Cl,vol)), a cAMP-dependent Cl(-) current (I(Cl,cAMP))) I(AB) was augmented by tefluthrin, fenpropathrin, alpha-cypermethrin (but not tetramethrin). I(AB) was insensitive to chlorotoxin, gadolinium and picrotoxin. Thus, I(AB) exhibits a distinct pharmacological profile from other known cardiac anion conductances.

This report describes a hitherto unreported anionic background current from human atrial cardiomyocytes. Under whole-cell patch-clamp with anion-selective conditions, an outwardly rectifying anion current (I(ANION)) was observed, which was larger with iodide than nitrate, and with nitrate than chloride as charge carrier. In contrast with a previously identified background anionic current from small mammal cardiomyocytes, I(ANION) was not augmented by the pyrethroid tefluthrin (10 microM); neither was it inhibited by hyperosmolar external solution nor by DIDS (200 microM); thus I(ANION) was not due to basal activity of volume-sensitive anion channels. I(ANION) was partially inhibited by the Cl(-) channel blockers NPPB (50 microM) and Gly H-101 (30 microM). Incorporation of I(ANION) into a human atrial action potential (AP) simulation led to depression of the AP plateau, accompanied by alterations to plateau inward calcium current, and to AP shortening at 50% but not 90% of complete repolarization, demonstrating that I(ANION) can influence the human atrial AP profile.

We investigated the difference in the characteristics of endothelin-1 (ET-1)-induced contraction and the responses of intracellular Ca(2+) concentration ([Ca(2+)](i)) between rat small pulmonary artery and renal artery. ET-1 (30 nM) failed to elicit any contraction in renal arteries pretreated with 3 microM BQ-123, an ETA blocker. However, in the pulmonary artery a combination of BQ-123 and BQ-788, an ETB blocker (5 microM each), only partially inhibited the ET-1-induced contraction (by 25%). To focus on the ETA receptor, in the presence of 5 microM BQ-788, nitric oxide donors (sodium nitroprusside and (+/-)-S-nitroso-N-acetylpenicillamine) and forskolin reduced both the ET-1-induced contraction and increase in [Ca(2+)](i) in both pulmonary and renal arteries. However, the effects were stronger in the renal than in the pulmonary artery. ET-1-induced increase in [Ca(2+)](i) was only partially attenuated by 10 microM verapamil (to 81% of control) in pulmonary arteries but was reduced to 56.1% of control in renal arteries. Our results provide evidence that ET-1 may activate ET receptor(s) insensitive to both BQ-123 and BQ-788 in rat small pulmonary artery, at least under these conditions. Furthermore, the effects of relaxants such as L-type Ca(2+) channel blocker and nitric oxide donors on the ET-1-induced contraction were studied.

Key elements of tumor development include proliferation, migration, invasiveness, and angiogenesis. Activation of the volume-sensitive organic osmolyte/anion channel (VSOAC) has been suggested to play a role in all of these processes. VSOACs may therefore represent an important therapeutic target in the etiology of cancer. However, pharmacological inhibitors of VSOAC are nonselective and of low potency, highlighting the importance of identifying novel regulators of the channel. The use of electrophysiological methods coupled with techniques such as pull-down assays, yeast 2-hybrid, and functional protein arrays have already proved valuable in studying protein-protein interactions in a variety of systems. Some of these methods have been used to identify small molecules that modulate the function of other types of ion channels. Given that several proteins have already been identified as putative modulators of VSOACs, proteomics technologies may prove useful in elucidating the molecular identity of VSOACs and helpful in identifying novel modulators of channel function. In this paper, we review the involvement of VSOACs in tumor development processes and its regulation by pharmacological agents and cellular proteins. Proteomic approaches to study protein-protein interactions and how such approaches may be used to study VSOACs are also discussed. We speculate on how modulation of protein-protein interactions may result in the identification of a novel class of compounds for modulating VSOACs.

CsCl has been shown to be arrhythmogenic in-vivo and to cause early afterdepolarizations (EADs) in isolated cardiac preparations, but the underlying electrophysiological mechanisms are ill-defined. To elucidate these actions further, the effects of extracellular solutions containing 3 mM CsCl and either 2 mM KCl (Cs2K solution) or 5 mM KCl (Cs5K solution) on membrane potential and ionic currents in rat and guinea-pig ventricular myocytes were compared. Cs2K solution rapidly and reversibly inhibited outward I(K1), and reduced other K(+) currents by about 20%. Current-clamped myocytes were rapidly hyperpolarized by this solution and action potentials were prolonged, but EADs were not observed. In contrast, EADs were triggered by E-4031, H(2)O(2), and the pyrethroid tefluthrin. Membrane-potential changes reversed after replacing Cs2K with Cs5K solution, with the recovery of 50% of outward I(K1). These results suggest that Cs2K solution inhibited I(K1) and caused a late prolongation of the action-potential duration, but the affected membrane potentials were too negative to elicit EAD mechanisms. In isolated hearts perfused with modified Tyrode's, Cs2K, and Cs5K solutions, bradycardia and arrhythmias were evoked by both CsCl-containing solutions. A comparison of such results with the effects of these solutions on myocytes suggests that I(K1) inhibition and EADs in ventricular myocytes are unlikely to be involved in arrhythmogenesis under our conditions.

Understanding the way in which single nucleotide polymorphisms and mutations in the human genome result in individual susceptibility to disease is a major goal in the postgenomic era. Such knowledge should accelerate the development of personalised medicine in which drug treatment can specifically match an individual's genotype. High-throughput DNA sequencing is generating the initial information required, but new technologies are required that can rapidly characterise the phenotypic effects of the identified polymorphisms. For example, many thousands of allelic variants of the p53 gene have been described and are responsible for more than 50% of cancers, however few of the protein products have been functionally characterised. Here we have quantified in parallel the effects of mutations and polymorphisms on the DNA-binding function of the p53 oncoprotein using a protein microarray, allowing their subclassification according to functional effect. Protein-protein interactions between p53 variants and (i) a regulatory oncoprotein, (ii) a regulatory kinase resulting in on-chip phosphorylation, are also described, suggesting the more general utility of this high-throughput assay format.

The acquired form of the long-QT syndrome (LQTS) is a major safety consideration for the development and subsequent use of both cardiac and non-cardiac drugs; it is usually associated with pharmacological inhibition of cardiac HERG-encoded potassium channels. Clomiphene is an anti-estrogen agent used extensively in the treatment of infertility and is not associated with a risk of QT interval prolongation, in contrast to a structurally related compound tamoxifen. We describe here a potent inhibitory effect (IC(50) = 0.18 microM) of clomiphene on HERG ionic current (I(HERG)) recorded from a mammalian cell line expressing HERG channels. Inhibition of I(HERG) by clomiphene showed voltage-dependence and developed quickly following membrane depolarisation, indicating contingency of block on HERG channel gating. At 100 nM, clomiphene and the related anti-estrogen tamoxifen produced similar levels of I(HERG) blockade (p > 0.05). Experiments on guinea-pig isolated perfused hearts revealed that, despite its inhibitory action on I(HERG), clomiphene produced no significant effect at 1 microM on uncorrected QT interval (p > 0.1) nor on rate-corrected QT interval (QT(c); p > 0.1 for QT(c) determined using Van de Water's formula). The disparity between clomiphene's potent I(HERG) inhibition and its lack of effect on the QT interval underscores the notion that I(HERG) pharmacology may best be used alongside other screening methods when investigating the QT-prolonging tendency and related cardiotoxicity of non-cardiac drugs.

1. There are no effective ways of screening for potential modulators of volume-regulated anion channels in their native cell type. Generally, cell lines are used for this purpose. Using HeLa and C6 glioma cells, we identified the pyrethroids as a novel class of compounds that inhibit taurine efflux through volume-regulated anion transport pathways in these cells. Subsequently, we examined their effects on volume-regulated anion channels in guinea-pig ventricular myocytes to determine whether results obtained using cell lines could be extrapolated to other tissues. 2. Tetramethrin inhibited taurine efflux in both HeLa and C6 glioma cells with Ki values of approximately 26 and 16 micro mol/L, respectively. Bioallethrin and fenpropathrin inhibited volume-sensitive taurine efflux from C6 glioma cells, but not from HeLa cells. The Ki values for bioallethrin and fenpropathrin were 70 and 59 micro mol/L, respectively. 3. Volume-sensitive I- efflux was observed in HeLa cells but not in C6 glioma cells, suggesting that the taurine efflux pathway in C6 glioma cells may be different to that of the I- efflux pathway. Cyfluthrin, tetramethrin, fenpropathrin, tefluthrin and bioallethrin all significantly inhibited volume-sensitive I- efflux from HeLa cells at 100 micro mol/L. 4. Patch-clamp experiments have shown inhibition of ICl,vol in guinea-pig ventricular myocytes by fenpropathrin, but not tetramethrin or cypermethrin, at 100 micro mol/L. This revealed that further differences exist between ICl,vol in guinea-pig ventricular myocytes and the anion transport pathways in C6 glioma and HeLa cells. 5. In conclusion, we have shown that pyrethroids differentially inhibit volume-regulated anion and taurine efflux in a number of cell types. Because these compounds have different effects in different cells, it is likely that: (i) more than one pathway is involved in the volume-sensitive transport of anions and organic osmolytes; and (ii) the molecular identities of the channels underlying anion transport are different. Finally, for the reasons given above, care should be taken when extrapolating data from one cell type to another. However, in the absence of an existing high-throughput screen, taurine efflux still represents a viable route for the identification of potential modulators of volume-regulated ion channels.

1. The effects of clomiphene (CLM) on cardiac outward K+ current components from rat isolated ventricular myocytes were investigated using the whole-cell patch-clamp technique. Clomiphene (10 micromol/L) significantly inhibited both peak (Ipeak) and end-pulse (Ilate) outward currents (elicited by a 500 msec voltage step from -40 to +50 mV in the presence of K+-containing intracellular and extracellular solutions) by approximately 37% (n = 6; P < 0.01) and 49% (n = 6; P < 0.01), respectively. In contrast, CLM had no effect on outward currents when K+-free solutions were used. 2. A double-pulse protocol and Boltzmann fitting were used to separate individual K+ current components on the basis of their voltage-dependent inactivation properties. At potentials positive to -80 mV, two inactivating transient outward components (Ito) and (IKx) and a non-inactivating steady state component (Iss) could be distinguished. 3. Clomiphene inhibited both Ito and Iss. The maximal block of Ito and Iss induced by CLM (100 micromol/L) was approximately 61% (n = 5) and 43% (n = 5) with IC50 values of 1.54 +/- 0.39 and 2.2 +/- 0.4 micromol/L, respectively. In contrast, the peak magnitude of IKx was unaltered by CLM, although its time-course of inactivation was accelerated. 4. Further experiments whereby myocytes were superfused with the vasoactive peptide endothelin (ET)-1 (20 nmol/L) revealed that CLM (10 micro mol/L) completely abolished the ET-1-sensitive component of Iss. 5. Our findings demonstrate, for the first time, the effects of CLM on distinct cardiac K+ current components and show that CLM modulates the voltage-gated K+ current components Ito and IKx and inhibits the steady state outward current Iss in rat ventricular myocytes.

FACS analysis and [14C]-taurine efflux were used to determine whether activation of the volume-sensitive organic osmolyte/anion channel plays a role in cell cycle progression. This was achieved by examining the effects of a collection of (i) H(1) antagonists and tricyclic antidepressants with a known inhibitory effect on cell cycle progression, and (ii) antidepressants and oestrogen receptor modulators with molecular structures likely to confer inhibition of the volume-sensitive organic osmolyte/anion channel. Of the 13 compounds examined in this study, the following showed no cytotoxicity following a 48-h exposure, and specifically inhibited osmosensitive taurine efflux (over lactate transport and anion exchange) with IC(50) values of (in microM): fluoxetine, approximately 14; fluvoxamine, approximately 24; amitriptyline, approximately 32; imipramine, approximately 32; mianserin, approximately 40. A 48-h application of these compounds at these concentrations significantly increased arrest in the G0/1 stage of the cell cycle by approximately 10%. The uniformity and specificity of the response elicited by these compounds strongly reinforces a correlation between cell cycle progression and osmosensitive taurine efflux activation.

The aim of this study was to determine the effects of the antiestrogen agent clomiphene on cardiac anionic and cationic sarcolemmal ion channels. Whole-cell recordings were made from rat and guinea pig ventricular myocytes. Clomiphene inhibited the volume-regulated chloride current [I(Cl,vol), activated by cell swelling after hypotonic shock (approximately 145 mOsM)] with an IC(50) value of approximately 9.4 microM. In contrast, at concentrations up to 100 microM, clomiphene failed to inhibit both the chloride current activated by cyclic AMP (I(Cl,cAMP)) and the anionic background current (I(AB)). At 10 microM, clomiphene blocked the voltage-gated fast sodium current and the L-type calcium current (I(Ca,L)) in both species. The voltage-independent fractional block of I(Ca,L) induced by clomiphene (10 microM) was approximately 82%, this concentration also inhibited the inwardly rectifying K(+) current with a fractional current block of approximately 26% at -90 mV. Fractional block of outward current at +70 mV in rat was approximately 25%, implying that delayed rectifying K(+) channels were also affected by clomiphene. We conclude that clomiphene shows selectivity for I(Cl,vol) over I(Cl,cAMP) and I(AB) and therefore represents a useful tool for studying chloride conductances in isolated ventricular myocytes with interfering currents blocked. However, due to its effects on cation conductances it would be of little value in this regard for other types of in vitro or in vivo experiments.

Here we describe for the first time a cell-based scintillation proximity assay using membrane soluble scintillants (MSS). MSS have a scintillant "head" group (2,5-diphenyloxazole) attached to a lipophilic "tail." MSS do not scintillate in an aqueous environment in the presence of a radioactive source: however, in a non-aqueous environment, such as a lipid bilayer (e.g., liposome or cell membrane), scintillation does occur. MSS can be incorporated into liposomes. When these MSS-containing liposomes are fused with the plasma membranes of cells in culture the MSS are incorporated into the cell membrane. Radiolabelled molecules in close proximity to the cell membrane will then elicit a scintillation signal. This system has been used to successfully monitor [(14)C]methionine uptake in HeLa cells and may be used in radiochemical and radioligand binding assays either in vivo or on microsomal preparations obtained from tissues. This new scintillation proximity technology could be readily adapted for high-throughput screening.

Objective: To determine whether, in native pulmonary arterial smooth muscle cells (PASMC), K(V)2.1 delayed-rectifying K(+) channels are central to the process of hypoxic pulmonary vasoconstriction., Methods: In this study, we tested for the presence of K(V)2.1 channel transcripts in rat small pulmonary arteries using RT-PCR, and for the protein itself using immunolocalisation. The contribution of K(V)2.1 channels to whole-cell K(V) currents (I(KV)) and their role in hypoxic inhibition of I(KV) in native PASMC was investigated utilising patch-clamp recordings., Results: K(V)2.1 mRNA expression and AbK(V)2.1 (anti-K(V)2.1 antibody) protein immunoreactivity were both present in small pulmonary arteries. Dialysis of PASMC with AbK(V)2.1 significantly attenuated I(KV) by 67% at +50 mV. Hypoxia ( approximately 20-30 mmHg) inhibited I(KV) by approximately 70% at +50 mV. Ablation of currents associated with K(V)2.1 using AbK(V)2.1 caused a marked reduction in the amplitude of I(KV). Hypoxia in the presence of the antibody did not affect the magnitude of I(KV)., Conclusions: These results indicate that K(V)2.1 channel subunits exist within small pulmonary arteries and conduct a significant part of I(KV) within native PASMC. Furthermore, application of AbK(V)2.1 abolishes hypoxic inhibition of I(KV) in native PASMC suggesting that K(V)2.1 channels play a pivotal role in mediating hypoxic pulmonary vasoconstriction.

Utilising the patch-clamp recording technique we have demonstrated for the first time the effects of hypoxia on the background current in pulmonary arterial endothelial cells. Electrophysiological studies revealed the presence of a novel oxygen-sensitive, non-selective cation conductance (I(NSC)) in these cells. The inward component of I(NSC) was significantly potentiated by hypoxia. Both the inward and outward components of I(NSC) were inhibited by both La(3+) and Gd(3+). Hypoxic activation of I(NSC) may provide an important Ca(2+) influx pathway essential for the release of a pulmonary-selective vasoconstrictor pivotal to the sustained phase of hypoxic pulmonary vasoconstriction.

This report describes for the first time a novel anionic background current (I(AB)) identified in guinea-pig isolated ventricular myocytes. It also shows that I(AB) has both novel and differential pharmacology from other (cardiac) chloride currents. Using the whole-cell patch-clamp technique and external anion substitution, I(AB) was found to be outwardly rectifying and highly permeable to NO(-)(3), with a relative permeability sequence of NO(-)(3) > I(-) > Cl(-). I(AB) was not blocked by 50 microM DIDS, by hypertonic external solution, or by the nonselective protein kinase inhibitor H7-DHC. Exposure to the pyrethroid agent tefluthrin (10 microM) increased the current density of I(AB) significantly at positive voltages (P < 0.05), but had no significant effect on other cardiac chloride currents. We conclude that I(AB) possesses a distinct pharmacology and does not fall into the three major classes of cardiac chloride conductance commonly reported., ((C)2002 Elsevier Science (USA).)

This study demonstrates for the first time that endothelial cells freshly isolated from small pulmonary arteries of the rat, based on their electrophysiological profile, possess two distinct populations of cells. Immunohistochemical staining revealed the presence of both anti-Kv1.5 and anti-Kir2.1 immunoreactivity in the endothelium of small pulmonary arteries. Patch-clamp studies demonstrated that 90% of cells studied exhibited an electrophysiological profile that was characterized by a delayed rectifier K+ conductance. However, the remaining 10% of cells studied showed the complete absence of a delayed rectifier K+ current and were characterized by an inward rectifier K+ conductance. Together these results indicate that endothelial cells isolated from rat small pulmonary arteries possess a heterogeneous population of cells that may be distinguished by their markedly different electrophysiological profiles. These different populations of cells may differ in their control of the resting membrane potential of endothelial cells, and thereby altering Ca2+ homeostasis and release of vasoactive compounds. These findings may therefore have important implications for understanding the regulation of pulmonary vascular tone.

Pyrethroid insecticides are known to modify neuronal sodium channels, inducing persistent, steady-state sodium current at depolarized membrane potentials. Cardiac myocytes are also rich in sodium channels but comparatively little is known about the effect of pyrethroids on the heart, or on the cardiac sodium channel isoform. In the present study therefore, we determined the actions of type I and type II pyrethroids against rat and guinea pig ventricular myocytes under current and voltage clamp, and on isolated perfused rat hearts. In myocytes, tefluthrin (type I) and fenpropathrin and alpha-cypermethrin (type II) prolonged action potentials and evoked afterdepolarizations. The time course of sodium current (I(Na)) was also prolonged by these compounds. Pyrethroids delayed I(Na) inactivation, when measured under selective conditions as current sensitive to 30 microM tetrodotoxin, by increasing the proportion of slowly inactivating current at the expense of fast inactivating current. Further experiments, focusing on fenpropathrin, revealed that its effects on I(Na) inactivation time course were dose-dependent, and the Na(+) "window-current" was increased in its presence. In unstimulated, isolated hearts perfused with the same pyrethroids, the variability in contraction amplitude increased due to variations in the intervals between heartbeats. These potentially arrhythmogenic changes are consistent with the effects observed at the cellular level. The type I pyrethroid tetramethrin had little effect in any of the preparations. These findings suggest that some pyrethroids possess considerable mammalian cardiac arrhythmogenic potential, the manifestation of which in vivo may depend on the route of exposure.

In conclusion, the meeting was viewed positively by academics, industrialists and administrators. The scientists generated considerable interest with some positive follow-up. Above all, it was clear that Bristol University is working to support the bridge that already spans the gap between academic and industrial pharmacology.

Hypoxic pulmonary vasoconstriction (HPV) is a mechanism whereby capillary perfusion is modulated to match alveolar ventilation by diverting blood flow away from poorly ventilated regions of the lung. K+ channels, sensitive to changes in oxygen tension, are thought to play a pivotal role in initiating contraction of pulmonary arterial smooth muscle cells. However, the specific channel subtypes involved have not yet been identified. Using RT-PCR, we have investigated the expression of delayed rectifying (Kv) channel mRNA in rat main and small pulmonary arteries and, for comparison, the systemic mesenteric artery. We have identified and fully sequenced a rat Kv9.2 cDNA and also demonstrated the presence of Kv1.7 and Kv4.1. The presence and relative distribution of Kv1.2, Kv1.5, Kv2.1, and Kv9 mRNA is consistent with the proposed contribution of these subunits to oxygen sensing by K channels, previously described in pulmonary arteries. Our data addresses the controversy relating to the likely distribution of Kv channels involved in oxygen sensing without necessarily implying that such subunits are directly responsible for this process. The differential expression of other subunits, particularly Kv4, indicates that these too may have a role in HPV, revealing the need for further biophysical evaluation of these channel subtypes.

Drugs that block cardiac cation channels have been marketed as the therapeutic answer to cardiac arrhythmia. However, such molecules have been only moderately successful at improving the survival of cardiac patients, and so new targets have been needed for future antiarrhythmic agents. This article outlines the properties and roles of Cl(-) channels, which are one of these new targets, and describes an approach for identifying novel CI(2) channel modulators.

Background: We describe a simple method using membrane potential ramps for rapidly determining the ion-channel selectivity of drugs that affect action-potential duration in isolated cardiac myocytes. The method allows the simultaneous assay of compounds on a number of ionic currents in a single cardiac cell., Methods: Trains of membrane potential ramps were applied from -90 to +70 mV at 0.33 Hz to obtain a consistent "signature current," in which the major individual currents involved in the cardiac action potential could be easily identified. Confirmatory experiments were performed using known inhibitors of these currents., Results: The identities of the currents in the signature were established by varying the concentrations of extracellular cations and by adding known ion channel blockers to superfusion solutions. Inhibition of each current had a characteristic and reproducible effect on the overall signature current., Conclusions: The consistent current signature in the presence and absence of blockers suggests that this method could be used for tertiary electrophysiological evaluation of compounds, eg, in a drug discovery program focusing on antiarrhythmic agents. The ability to assay for secondary effects of novel compounds against multiple currents in the target cell type is convenient and avoids the artefacts associated with using artificial expression systems.

The electophysiological effects of endothelin-1 (ET-1) and their relationship to contraction remain unclear in the renal circulation. Using endotheliumdenuded arteries from the main branch of the renal artery proximal to the kidney of the rat, we have examined its effects on tension and conducted parallel patch-clamp measurements using freshly isolated smooth muscle cells from this tissue. Pharmacological experiments revealed that ET-1 produced constriction of renal arteries dependent on the influx of extracellular Ca(2+), mediated solely through ET(A) receptor stimulation. Current-clamp experiments revealed that renal arterial myocytes had a resting membrane potential of approximately 32 mV, with the majority of cells exhibiting spontaneous transient hyperpolarizations (STHPs). Application of ET-1 produced depolarization and in those cells exhibiting STHPs, either caused their inhibition or made them occur regularly. Under voltage-clamp conditions cells were observed to exhibit spontaneous transient outward currents (STOCs) inhibited by iberiotoxin. Application of voltage-ramps revealed an outward current activated at approximately -30 mV, sensitive to both 4-AP and TEA. Taken together these results suggest that renal arterial myocytes possess both delayed rectifying K(+) (K(V)) and Ca(2+)-activated K(+) (BK(Ca)) channels. Under voltage-clamp, ET-1 attenuated the outward current and reduced the magnitude and incidence of STOCs: effects mediated solely as a consequence of ET(A) receptor stimulation. Thus, in conclusion, activation of ET(A) receptors by ET-1 causes inhibition of K(V) and BK(Ca) channel activity, which could promote and/or maintain membrane depolarization. This effect is likely to favour L-type Ca(2+) channel activity providing an influx pathway for extracellular Ca(2+) essential for contraction.

Effects of extracellular anions were studied in electrophysiological experiments on freshly isolated rat ventricular myocytes. Under current-clamp, action potential duration (APD) was prolonged by reducing the extracellular Cl(-) concentration and shortened by replacement of extracellular Cl(-) with I(-). Under voltage-clamp, membrane potential steps or ramps evoked an anionic background current (I(AB)) carried by either Cl(-), Br(-), I(-) or NO(3)(-). Activation of I(AB) was Ca(2+)- and cyclic AMP-independent, and was unaffected by cell shrinkage. I(AB) was insensitive to stilbene and fenamate anion transport blockers at concentrations that inhibit Ca(2+)-, cyclic AMP- and swelling-activated Cl(-) currents in ventricular cells of other mammals. These results suggest that I(AB) may be carried by a novel class of Cl(-) channel. Correlation of anion substitution experiments on membrane current and action potentials revealed that I(AB) could play a major role in controlling rat ventricular APD. These findings have important implications for those studying cardiac Cl(-) channels as potential targets for novel antiarrythmic agents.

We have, for the first time, developed a reliable method for freshly isolating viable endothelial cells from resistance-sized rat pulmonary arteries. The endothelial origin of these cells was confirmed using indirect immunofluorescence, utilizing fluorescently labeled low-density lipoprotein. Biophysical and pharmacological patch-clamp experiments conducted under quasiphysiological cationic gradients revealed that these cells had a mean resting membrane potential of approximately -38 mV and displayed a delayed-rectifying K(+) current. Immunohistochemical staining of rat lung cross-sections revealed an abundance of K(V)1.5 channel protein in pulmonary endothelium. This is the first report of a delayed-rectifying K(+) current in endothelial cells of resistance-sized pulmonary arteries. Since changes in membrane potential associated with K(+) channel activity affect release of vasoactive substances from endothelial cells, this finding has important implications for understanding the mechanisms underlying control of pulmonary vascular tone., (Copyright 1999 Academic Press.)

We investigated the effects of the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) on endothelin-1 (ET-1)-induced constriction of rat small pulmonary arteries (diameter 100-400 microm) in vitro, following endothelium removal. ET-1 (30 nM) induced a sustained constriction of rat pulmonary arteries in physiological salt solution. Arteries preconstricted with ET-1 were relaxed by niflumic acid (IC50: 35.8 microM) and NPPB (IC50: 21.1 microM) in a reversible and concentration-dependent manner. However, at concentrations known to block Ca++-activated Cl- channels, DIDS (

Uridine nucleotides are known to cause constriction of pulmonary arterial smooth muscle. However, the P2 receptor subtypes underlying the contractile effects of these nucleotides in the pulmonary circulation have not been determined. We have used myography and the patch-clamp recording technique to compare the effects of UTP and UDP in isolated small pulmonary arteries (diameter 100 to 400 microm) and their constituent smooth muscle cells. In endothelium-denuded arteries, both UTP and UDP (0.01 to 3 mmol/L) induced concentration-dependent increases in tension that were independent of P2X receptor stimulation. The UDP-mediated increase in tension was significantly less sensitive to the nonselective P2 receptor blocker suramin than the UTP-mediated increase in tension. In single isolated arterial myocytes, voltage-clamped at -50 mV (close to the resting membrane potential of these cells), application of both UTP and UDP evoked periodic oscillations of inward current primarily because of a Ca2+-activated Cl- current (ICl,Ca). Oscillations of ICl,Ca evoked by UTP were reversibly inhibited by suramin, although those evoked by UDP were insensitive to the antagonist. In addition to confirming the presence of classical P2Y2 receptors, these results also provide functional evidence for the existence of a novel UDP receptor in pulmonary arterial myocytes, which may contribute to pyrimidine-evoked vasoconstriction. This notion is supported by molecular evidence that demonstrates the presence of P2Y6 receptor transcripts in rat pulmonary arterial smooth muscle.

Hypoxic pulmonary vasoconstriction (HPV) may be mediated, in part, by an oxygen-sensing mechanism intrinsic to pulmonary arterial smooth muscle. It has been proposed that hypoxia inhibits a K+ conductance, which promotes membrane depolarization, subsequent activation of L-type Ca2+ channels and ultimately constriction. We have monitored hypoxia-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) of single myocytes isolated from the rat pulmonary arterial tree using microspectrofluorimetry and ratiometric measurement of indo-1 fluorescence. The basal level of [Ca2+]i was in the range 35-80 nM and cells were quiescent at rest exhibiting no spontaneous oscillations in [Ca2+]i. When the extracellular K+ concentration ([K+]o) was raised to 20 mM, the [Ca2+]i increased from approximately 60 to approximately 100 nM. This increase was abolished by nifedipine, demonstrating the presence, and need for activation, of functional voltage-gated L-type Ca2+ channels. Hypoxia (PO2 < or = 30 mmHg; throughout) had little effect on the resting [Ca2+]i in myocytes isolated from either the main intrapulmonary artery, or its primary, secondary or tertiary branches. However, upon raising the [Ca2+]i by increasing [K+]o to 20 mM, hypoxia was found to lower [Ca2+]i from approximately 110 to approximately 70 nM, in cells isolated from the main conduit and primary branches of the intrapulmonary artery. In marked contrast, when [Ca2+]i was raised, by increasing [K+]o to 20 mM, in myocytes isolated from secondary and tertiary branches of the intrapulmonary artery, hypoxia induced a further reversible increase in the [Ca2+]i from approximately 160 to approximately 240 nM. Neither hypoxia alone nor in combination with 20 mM K(+)o induced any increase in the [Ca2+]i in the presence of nifedipine. We conclude that hypoxia may modulate [Ca2+]i in rat pulmonary artery myocytes only following its elevation by a depolarizing stimulus.

The electrophysiological effects of endothelin (ET)-1 were compared in myocytes isolated from rat small pulmonary artery, basilar artery and aorta. ET-1 evoked depolarization in all three smooth muscle cell types. Depolarizing oscillations in membrane current also were observed in pulmonary and aortic myocytes. In voltage-clamp experiments ET-1 induced a gradual inhibition of the Ca(++)-independent outward current (IK) in pulmonary and aortic myocytes, whereas in basilar myocytes ET-1 inhibited the Ca(++)-activated K+ current (IK(Ca)). ET-1 also evoked a transient enhancement of IK(Ca) and oscillations in inward current in aortic and pulmonary myocytes. The inward currents were inhibited by caffeine, which suggests Ca(++)-dependent activation. Ion-exchange experiments indicated that in pulmonary myocytes oscillatory currents were caused solely by the movement of Cl-, whereas in aortic myocytes they were the consequence of both Ca(++)-activated Cl-(ICl(Ca)) and non-selective cation currents (INS). No inward current was evoked in basilar myocytes in response to ET-1 or photorelease of Ca++, which suggests that these cells do not possess ICl(Ca). Experiments with ET receptor ligands indicated that in basilar myocytes ETA receptor stimulation is responsible for IK(Ca) inhibition, whereas in aortic and pulmonary myocytes ETB and ETA receptor stimulation mediates inhibition of IK and activation of ICl(Ca), INS and IK(Ca), respectively. In the future, it may be possible to exploit these differential effects of ET-1 pharmacologically to assist development of tissue-specific modulators for the treatment of vascular disease.

Sarafotoxin S6c [STXS6c; a selective endothelin-B (ETB) receptor agonist] causes constriction of isolated pulmonary arteries. In perforated-patch experiments on pulmonary arterial myocytes, ET-1 and STXS6c induced a gradual inhibition of the delayed rectifier K current (IKV), the profile of which resembled that carried by Kv1.5. Reverse-transcriptase polymerase chain reaction (RT-PCR) experiments revealed mRNA encoding this channel, and immunolocalization experiments demonstrated expression of the channel protein in pulmonary arterial smooth muscle. It is tempting to speculate that ETB receptor coupling to Kv1.5 may be implicated in contraction after stimulation of these receptors.

Pulmonary arteries constrict in response to hypoxia, a process thought to involve oxygen sensing by K+ channels. We therefore investigated the effects of hypoxia on voltage-activated K+ currents in myocytes isolated from rat small pulmonary arteries using the patch-clamp recording technique. Experiments with iberiotoxin and intracellularly applied Ca2+ chelating agents revealed that hypoxia (PO2, 20-30 mmHg; throughout) inhibited the Ca(2+)-insensitive component of the delayed voltage-activated outward K+ current. Hypoxia did not affect the membrane potential of these cells until they were depolarized by extracellular application of 20 mM K+, current injection or endothelin-1. Hypoxia caused little depolarization in the presence of prostaglandin F2 alpha, an agonist which was ineffective at inducing depolarization. These results suggest that an initial 'priming' depolarization may confer a sensitivity to hypoxia by activating delayed rectifier (Kv) channels. Once active, these channels can then be closed by hypoxia, leading to further depolarization. It is unlikely, therefore, that Kv channels are involved in controlling the resting membrane potential of these cells.

Neither the electrophysiological effects of purinergic receptor stimulation nor the role of ATP in regulating the tone of pulmonary arterial smooth muscle has been determined. Therefore, we investigated the effects of purine nucleotides on acutely dissociated smooth muscle cells from rat small pulmonary arteries using the patch-clamp recording technique. Extracellular application of ATP activated a fast transient inward current (which decayed in the continued presence of the nucleotide) and produced sustained periodic oscillations of predominantly inward current. Pharmacological and anion substitution experiments revealed that the transient inward current was carried by the movement of cations. In contrast, the periodic oscillations of current were due primarily to a Ca(2+)-activated Cl- current (ICl,Ca) dependent on the release of Ca2+ from intracellular stores. Experiments using ATP analogues revealed the following order of potency for activation of the fast transient inward current: 2-methylthio ATP (2-meSATP) > ATP > alpha,beta-methylene ATP (alpha,beta-meATP) > > ADP > UTP = adenosine. Cross desensitization was seen between applications of ATP, alpha,beta-meATP, and 2-meSATP, suggesting that these agonists act via a common site. The order of potency for activation of ICl,Ca was UTP = ATP > > ADP > or = 2-meSATP > alpha,beta-meATP = adenosine. Both the fast transient inward current and ICl,Ca evoked by ATP and its analogues were abolished by the nonselective P2 purinoceptor antagonist suramin. These results show the existence of P2x and P2U purinoceptor subtypes in pulmonary arterial smooth muscle cells. Stimulation of these receptors results in activation of a fast transient inward cation current and ICl,Ca, respectively. It is likely that ATP acts via these receptor subtypes to regulate pulmonary arterial tone under physiological or pathological conditions.

Endothelin (endothelin)-1 may in important role in the control of pulmonary arterial tone, part of its action being due to a putative ability to regulate membrane ion channel activity. Consequently, we have examined its effects on membrane currents in pulmonary arterial myocytes by using the patchclamp recording technique. Endothelin-1 (0.1-50 nM) activated an oscillatory inward current and caused a brief enhancement, followed by inhibition of the outward K+ current. Pharmacological and anion substitution experiments revealed that the oscillations of inward current were due to ET(A) receptor stimulation and subsequent activation of Ca(+2)-activated Cl- channels (EC50 > or = 16 nM). Enhancement of K+ current was inhibited by FR139317 (an ET(A) receptor antagonist) and by buffering intracellular Ca+2, which suggests that ET(A) receptor stimulation also activates Ca(+2)-activated K+ channels. Inhibition of K+ current (IC50 approximately 0.5 nM) by endothelin-1 was not dependent on phosphorylation or intracellular Ca+2 and was unaffected by FR139317 but was mimicked by ET(B) receptor agonists. It appears, therefore, that ET(A) and ET(B) receptors in pulmonary arterial myocytes are differentially coupled to Cl- and K+ channels. Modulation of these channels by endothelin-1 may prove to play an important role in regulating pulmonary arterial smooth muscle tone under both physiological and pathological conditions.

Simultaneous patch-clamp and Ca2+ fluorescence measurements have revealed depolarising oscillations in the membrane potential of arterial (pulmonary) myocytes in response to adenosine 5'-triphosphate (ATP) and endothelin-1 (ET-1). These oscillations (i) are due to the preferential activation of Ca(2+)-activated Cl-, over K+ currents, (ii) occur through a mechanism involving Ca2+ release from intracellular Ca2+ stores and (iii) are likely to promote constriction. These results provide a novel perspective into the relative contribution and importance of Ca2+ activated Cl- and K+ channels in controlling membrane potential of arterial smooth muscle in response to contractile agonists.

Large conductance Ca(2+)-activated K+ (Kca) channels are known to be activated by phosphorylation through cAMP- and cGMP-dependent kinase activation. In pulmonary arterial smooth muscle KCa channels are directly activated by ATP (but not by non-hydrolysable analogues) independently of the presence of cyclic nucleotides or the catalytic subunits of protein kinases. This study was designed to determine whether direct activation of KCa channels by ATP is apparent in other types of arterial smooth muscle. KCa channels of similar conductance to those of rat pulmonary artery (approximately 250 pS) were found in membrane patches excised from isolated smooth muscle cells from rat aorta, mesenteric and basilar arteries. In myocytes isolated from each of these arteries, intracellular application of ATP (in the absence of exogenous cyclic nucleotides or catalytic subunits) reversibly increased the open state probability of KCa channels: a response markedly reduced by a specific inhibitor of protein kinase A. Nucleotide sequence analysis of KCa channels revealed no homology with the majority of protein kinases. It is concluded that phosphorylation of KCa channels through the activity of a membrane tethered kinase related to protein kinase A (but lacking its regulatory subunits) may play an important role in controlling K+ flux in a range of arterial smooth muscle cell types.

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.

In contrast to systemic arteries those of the pulmonary circulation constrict in response to hypoxia: a mechanism known as "hypoxic pulmonary vasoconstriction" (HPV). It is has been established that changes in alveolar oxygen tension are the primary stimulus for triggering HPV. The mechanisms underlying this unique response are unknown. Neither the role of the endothelium nor that of the smooth muscle of the pulmonary arteries in oxygen sensing has been elucidated, although both tissues may be involved. Evidence is accumulating to suggest that K+ channels located in the plasma membranes of pulmonary arterial smooth muscle cells may play an important role in oxygen sensing. This process may involve novel mechanisms of signal transduction and K+ channel regulation by charged intermediates, redox reactions or membrane-delimited factors.

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.

Intracellular photorelease of Ca2+ demonstrated the presence of Ca(2+)-activated K+ channels in smooth muscle cells isolated from different locations of the rat pulmonary arterial tree. However, cell-free patch studies revealed marked differences in K+ channel distribution. In the main pulmonary artery the most frequently observed K+ channel was a approximately 245pS conductance Ca(2+)- and ATP-activated (KCa,ATP) channel. In small pulmonary arteries two K+ channel types predominated: the KCa,ATP channel and a approximately 185pS conductance K+ channel insensitive to intracellular Ca2+, ATP and voltage. This difference in K+ channel distribution may highlight a more complex regulatory mechanism for controlling membrane potential in small pulmonary arteries, reflecting their physiologically more important role in governing pulmonary vascular reactivity.

A novel class of Ca(2+)-activated K+ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these "KCa,ATP" channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of KCa,ATP channels to voltage, intracellular Ca2+ ([Ca2+]i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K+ and are approximately 20 times more selective for K+ ions than Na+ ions, with a K+ permeability (PK) of 4.6 x 10(-13) cm s-1.Ca2+ ions applied to the intracellular membrane surface of KCa,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca2+ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of KCa,ATP channels by Mg-ATP has an EC50 of approximately 50 microM. Although the EC50 is unaffected by [Ca2+]i, channel activation by Mg-ATP is enhanced by increasing [Ca2+]i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of KCa,ATP channels to Ca2+. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of KCa,ATP channels to Ca2+ and therefore voltage is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Using the patch-clamp recording technique, we have investigated the effects of chronic intracellular application of guanosine thiotriphosphate (GTP gamma S) by cell dialysis, on the potentiation of L-type Ca2+ currents (ICa) by isoprenaline and forskolin and also by GTP gamma S and cyclic AMP released intracellularly by flash-photolysis of their caged derivatives. 2. GTP gamma S prevented enhancement of ICa by isoprenaline with an IC50 of approximately 10 microM and considerably reduced the ability of forskolin to increase ICa. In addition GTP gamma S also reduced the time-to-peak response for potentiation of ICa by forskolin. Responses to forskolin were abolished by co-dialysis of cells with the cyclic AMP antagonist, Rp-adenosine-3'-5'-mono-thionophosphate (Rp-cAMPS). 3. Photoreleased GTP gamma S (PR-GTP gamma S; approximately 23 microM) generally induced a biphasic increase in ICa. This response was also inhibited by chronic intracellular dialysis with GTP gamma S with an IC50 of approximately 1 microM. 4. Pretreatment of cells with pertussis toxin (PTX) reversed the inhibitory effect of 100 microM GTP gamma S on isoprenaline-induced stimulation of ICa. However, PTX pretreatment did not restore the activating action of PR-GTP gamma S inhibited by chronic application of GTP gamma S. 5. Photoreleased cyclic AMP (approximately 5 microM; PR-cyclic AMP) increased peak ICa. This effect was inhibited by dialysis of cells with Rp-cAMPS and by stimulation of ICa by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Co-dialysis of cells with uncaged GTP gamma S reduced the time-to-peak for PR-cyclic AMP mediated activation of ICa but did not affect the magnitude of the response. 6. It is concluded that chronically applied GTP gamma S can (i) inhibit activation of ICa by isoprenaline by interacting with a PTX-sensitive guanosine nucleotide binding (G-) protein located upstream of adenylate cyclase (possibly Gi) and (ii) accelerate the response to cyclic AMP dependent phosphorylation possibly by interacting with a G-protein coupled directly to the channel. 7. In view of this diverse range of effects, care should be taken when using GTP gamma S to characterize G-protein-mediated events, since the resulting physiological response may be due to activation of several G-protein containing pathways.

1. Single guinea-pig ventricular myocytes were voltage clamped using the whole-cell configuration of the patch-clamp technique and membrane current generated by the Na(+)-Ca2+ exchange mechanism recorded. 2. Rapid increases in cytosolic free calcium ([Ca2+]i) evoked by flash photolysis of either nitr-5 or DM-nitrophen resulted in current relaxations, arising from a redistribution of exchanger carrier conformations induced by the changes in [Ca2+]i. 3. Relaxation time constants were temperature dependent with a temperature coefficient over a 10 degrees C range (Q10) of approximately 3 and also voltage dependent, decreasing on hyperpolarization for membrane potentials in the range +40 to -80 mV. 4. The experimental results are consistent with consecutive exchange models having electrogenic Na+ translocation steps, together with a site density and turnover rate similar to that for the Na(+)-K+ pump.

L-type calcium currents (ICa) were recorded from isolated ventricular myocytes by using standard patch-clamp methods. In the absence of agonist, photorelease of GTP by flash photolysis of intracellularly applied caged-GTP rapidly increased the amplitude of ICa over a wide range of membrane potentials. Control experiments clearly demonstrated that this effect was not due to either the release of photolytic by-products or to the light flash itself. The timecourse for activation of ICa by photolysis of caged-GTP was markedly altered by intracellular application of either GDP beta S or GTP gamma S. Upon maximal stimulation of ICa by intracellular dialysis with cAMP, photoreleased GTP induced a small, rapid increase in ICa followed by a gradual inhibition. The presence of Rp-cAMPS intracellularly reduced both the magnitude of the response to photoreleased GTP and its time to peak. Similar effects were observed when protein kinase inhibitor dialysed the cell interior, suggesting that both cAMP-dependent and independent processes were involved in this effect. We conclude that rapid release of GTP within ventricular myocytes, in the absence of agonist, causes rapid activation of L-type Ca2+ current. Mechanisms underlying this effect include stimulation of adenylate cyclase, together with other, as yet uncharacterized, GTP-dependent pathways for increasing ICa in the heart.

1. Patch-clamp recording techniques were used, to examine the effects of diazoxide on KATP currents in CRI-G1 insulin-secreting cells in the presence of non-hydrolysable nucleotides. 2. In the presence of non- or slowly-hydrolyzed ATP analogues, bathing the intracellular aspect of cell-free membrane patches diazoxide inhibited KATP channel activity. 3. Under whole-cell recording conditions, with various non-hydrolysable nucleotides present intracellularly (after dialysis), diazoxide induced KATP current activation. The largest activation occurred with Mg-adenylyl-(beta, gamma-methylene) diphosphate (Mg-AMP-PCP) present in the dialysing solution. This activation was diazoxide- and nucleotide-concentration-dependent. 4. In the absence of Mg2+, or in the presence of manganese (Mn2+) ions intracellularly, diazoxide did not induce KATP current activation, regardless of the species of nucleotide present in the pipette. 5. Intracellularly applied trypsin prevented the activation of KATP currents by diazoxide in the presence of Mg-AMP-PCP, an effect reversed by co-application of intracellular polymethylsulphonyl fluoride with the trypsin. 6. The application, by dialysis, of a CRI-G1 cell lysate, with negligible Mg-ATP, resulted in a substantial activation of the KATP current by diazoxide. 7. It is concluded that diazoxide can activate KATP channel currents by two separate pathways, one requiring a phosphorylation process, the other the presence of an intracellular protein coupled with a Mg-purine nucleotide.

1. We show that cromakalin and diazoxide, drugs that activate ATP-sensitive potassium (KATP) channels, abolish hypoxic pulmonary vasoconstriction (HPV) of isolated, perfused rat lungs. 2. Glibenclamide, an inhibitor of these channels, does not affect HPV, but it reverses the relaxation caused by cromakalim and diazoxide. 3. Tolbutamide, which has effects similar to glibenclamide in other tissues, paradoxically abolishes HPV, an effect reversed by glibenclamide. 4. These results suggest that: (i) pulmonary vessels contain KATP channels which are normally closed and are not opened by levels of hypoxia that cause constriction, (ii) tolbutamide acts on the pulmonary vasculature by a mechanism which differs from that of glibenclamide.

We have observed a novel class of calcium-activated potassium channel which is activated by physiological levels of intracellular ATP. These KCa,ATP channels are found on smooth muscle cells isolated from the pulmonary artery. Since their activation by ATP is Mg2+ dependent and is poorly evoked by non- or slowly-hydrolyzed ATP analogues, we conclude that it involves phosphorylation. We suggest that in hypoxia a reduction of intracellular ATP may reduce KCa,ATP channel activity and thereby tend to depolarize the cells. This effect would increase Ca2+ entry through voltage-activated Ca2+ channels and contribute to vasoconstriction.

L-type calcium currents were recorded from isolated ventricular myocytes using standard patch-clamp methods. Rapid release of photolabile guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), tetralithium salt, by flash photolysis of intracellular caged-GTP gamma S increased the magnitude of the L-type calcium current: an effect independent of the ultraviolet flash per se or the production of photolytic by-products. This increase in calcium current was markedly reduced by intracellularly applied guanosine 5-O-(thiodiphosphate), trilithium salt or by an excess of GTP gamma S. It is therefore likely that rapid release of GTP gamma S intracellularly in the absence of an agonist can, via a G protein-mediated mechanism, cause L-type calcium current activation. In the presence of Rp-adenosine 3,5-mono-thionophosphate (Rp-cAMP), photoreleased GTP gamma S results in a transient and much reduced increase in the amplitude of the L-type calcium current. We conclude that activation of Gs coupled to adenylate cyclase and ultimately cAMP-dependent phosphorylation may be primarily responsible for L-type channel activation, although a fast membrane-delimited (direct) pathway, not involving cytoplasmic second messengers, may also contribute to this effect.

1. Patch-clamp recording techniques were used to examine the effects of barbiturates upon the ATP-K+ channel, and voltage-activated channels present in the plasma membrane of CRI-G1 insulin-secreting cells. 2. Thiopentone inhibited ATP-K+ channel activity when applied to cell-attached patches or the intracellular or extracellular surface of cell-free patches. Secobarbitone and pentobarbitone were also effective inhibitors of ATP-K+ channels in cell-free patches, whereas phenobarbitone was ineffective. 3. The diabetogenic agent, alloxan, which is structurally related to the barbiturates also produced an inhibition of ATP-K+ channel activity in outside-out patches. 4. Whole-cell ATP-K+ currents were used to quantify the effects of the barbiturates: concentration-inhibition curves for thiopentone, secobarbitone and pentobarbitone resulted in IC50 values of 62, 250 and 360 microM respectively. Phenobarbitone at a concentration of 1 mM was virtually ineffective. 5. Calculation of the apparent membrane concentrations for these drugs indicate that for a given degree of ATP-K+ channel inhibition a similar concentration of each barbiturate is present in the membrane. This suggests that hydrophobicity plays a primary role in their mechanism of action. The pH-dependence and additive nature of barbiturate block also indicates a membrane site of action. 6. Thiopentone, (100 microM) was also found to inhibit differentially voltage-activated whole-cell currents. The relative potency of thiopentone at this concentration was 0.64, 0.38 and 0.12 for inhibiting Ca2+, K+ and Na+ currents respectively when compared with its ability to inhibit the ATP-K+ channel.