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1.
Biochem Biophys Res Commun ; 374(4): 742-6, 2008 Oct 03.
Article in English | MEDLINE | ID: mdl-18671941

ABSTRACT

Since, in addition to its growth-promoting actions, insulin-like growth factor-I (IGF-I) has rapid vasoactive actions, we investigated the effects of IGF-I on whole-cell ATP-sensitive K(+) (K(ATP)) currents of rat mesenteric arterial smooth muscle cells. IGF-I (10 or 30 nM) reduced K(ATP) currents activated by pinacidil or a membrane permeant cAMP analogue. Inhibition of phospholipase C, protein kinase C, protein kinase A, mitogen-activated protein kinase or mammalian target of rapamycin (mTOR) did not prevent the action of IGF-I. However, inhibition of K(ATP) currents by IGF-I was abolished by the tyrosine kinase inhibitor genistein or the phosphoinositide 3-kinase inhibitors, LY 294002 and wortmannin. Intracellular application of either phosphatidylinositol 4,5-bisphosphate (PIP(2)) or phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) increased the K(ATP) current activated by pinacidil and abolished the inhibitory effect of IGF-I. Thus, we show regulation of arterial K(ATP) channels by polyphosphoinositides and report for the first time that IGF-I inhibits these channels via a phosphoinositide 3-kinase-dependent pathway.


Subject(s)
Arteries/metabolism , Insulin-Like Growth Factor I/physiology , KATP Channels/antagonists & inhibitors , Myocytes, Smooth Muscle/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Androstadienes/pharmacology , Animals , Arteries/drug effects , Arteries/enzymology , Chromones/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Genistein/pharmacology , Insulin-Like Growth Factor I/pharmacology , KATP Channels/metabolism , Male , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Morpholines/pharmacology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/enzymology , Phosphatidylinositol 4,5-Diphosphate/pharmacology , Phosphatidylinositol Phosphates/pharmacology , Phosphoinositide Phospholipase C/antagonists & inhibitors , Phosphoinositide Phospholipase C/metabolism , Phosphoinositide-3 Kinase Inhibitors , Pinacidil/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Rats , Rats, Wistar , TOR Serine-Threonine Kinases , Wortmannin
2.
J Physiol ; 581(Pt 2): 479-93, 2007 Jun 01.
Article in English | MEDLINE | ID: mdl-17363390

ABSTRACT

The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells. hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating cellular excitability. In this study, we investigated muscarinic modulation of hERG currents and direct phosphorylation of channel subunits expressed in HEK-293 cells at physiologically relevant temperatures by protein kinase C (PKC). Activation of G(alpha q/11)-coupled M(3)-muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by either acute inhibition of PKC with 300 nm bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 h pretreatment of cells with phorbol 12-myristate 13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetylglycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [(32)P]orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of hERG.


Subject(s)
Ether-A-Go-Go Potassium Channels/metabolism , Ion Channel Gating , Potassium/metabolism , Protein Kinase C/metabolism , Receptor, Muscarinic M3/metabolism , Signal Transduction , Calcium/metabolism , Cell Line , Diglycerides/pharmacology , ERG1 Potassium Channel , Enzyme Activators/pharmacology , Ether-A-Go-Go Potassium Channels/drug effects , Ether-A-Go-Go Potassium Channels/genetics , Humans , Indoles/pharmacology , Ion Channel Gating/drug effects , Ionomycin/pharmacology , Ionophores/pharmacology , Maleimides/pharmacology , Membrane Potentials , Methacholine Chloride/pharmacology , Muscarinic Agonists/pharmacology , Mutation , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Receptor, Muscarinic M3/agonists , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Transfection
3.
J Physiol ; 575(Pt 3): 833-44, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16825302

ABSTRACT

Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage-activated K(+) (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin-1 (ET-1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose-free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC-IP), and Kv currents were further reduced in 10 mm glucose. In current-clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mm d-glucose, 10 nm ET-1 decreased peak Kv current amplitude at +60 mV from 23.5 +/- 3.3 to 12.1 +/- 3.1 pA pF(-1) (n = 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET-1 was prevented by PKC-IP. When d-glucose was increased to 10 mm, ET-1 no longer inhibited Kv current (n = 6). Glucose metabolism was required for prevention of ET-1 inhibition of Kv currents, since fructose mimicked the effects of d-glucose, while l-glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mm d-glucose, since pinacidil-activated ATP-dependent K(+) (K(ATP)) currents were reduced by 10 nm ET-1. This inhibition was nearly abolished by PKC-IP, indicating that endothelin receptors could still activate PKC in 10 mm d-glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.


Subject(s)
Endothelin-1/pharmacology , Glucose/pharmacology , Muscle, Smooth, Vascular/drug effects , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/drug effects , Vasoconstrictor Agents/pharmacology , Animals , Glucose/metabolism , Hyperglycemia/metabolism , In Vitro Techniques , Ion Channel Gating , Male , Membrane Potentials , Mesenteric Arteries/drug effects , Mesenteric Arteries/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Potassium Channels, Voltage-Gated/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Wistar
4.
J Pharmacol Toxicol Methods ; 54(2): 173-82, 2006.
Article in English | MEDLINE | ID: mdl-16632384

ABSTRACT

INTRODUCTION: Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. MATERIALS AND METHODS: We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. RESULTS: Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6+/-13.5 vs. 296.2+/-16.2 ms; p<0.01). Despite this effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically or with a patch electrode (91+/-46 vs. 81+/-20 nM). DISCUSSION: These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.


Subject(s)
Action Potentials/drug effects , Membrane Potentials/drug effects , Myocytes, Cardiac/drug effects , Animals , Artifacts , Calibration , Coloring Agents , Drug Evaluation, Preclinical , Guinea Pigs , In Vitro Techniques , Male , Microscopy, Confocal , Potentiometry , Pyridinium Compounds/pharmacology , Radiometry
5.
J Mol Cell Cardiol ; 39(4): 637-46, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16099467

ABSTRACT

ATP-sensitive K(+) (K(ATP)) channels are involved in ischemic cardioprotection induced by preconditioning (IPC), though the relative role of sarcolemmal (sK(ATP)) and mitochondrial (mitoK(ATP)) channels remains controversial. The sK(ATP)-selective sulphonylthiourea HMR 1098 has often been reported to be without effect on ischemic cardioprotection, suggesting minimal involvement of sK(ATP). Since some sulphonylureas show reduced potency under conditions of metabolic stress, we used patch clamp to assess the ability of HMR 1098 to block sK(ATP) currents of adult rat ventricular myocytes activated by metabolic inhibition (MI, NaCN+iodoacetate). In contrast to the prototype sulphonylurea glibenclamide, HMR 1098 (10 muM) was without effect on sK(ATP) currents, and also did not inhibit MI-induced action potential shortening. However, HMR 1098 blocked sK(ATP) current induced by the K(ATP) opener pinacidil (IC(50)=0.36+/-0.02 muM), and reversed pinacidil-induced action potential shortening. In inside-out patches, block by HMR 1098 was relieved by increasing MgADP concentrations (1-100 muM). HMR 1098 inhibited pinacidil-activated recombinant Kir6.2/SUR2A channels with a similar IC(50) (0.30+/-0.04 muM), but was less effective when channels were activated by low intracellular ATP. HMR 1098 displaced binding of the pinacidil analogue [(3)H]P1075 to native cardiac membranes with a biphasic inhibition curve. Our results show that HMR 1098 becomes a much less effective inhibitor of sK(ATP) during metabolic stress, and suggest that the lack of effect of HMR 1098 on ischemic cardioprotection reported in some studies may represent loss of block by the drug under these conditions rather than a lack of involvement of sK(ATP) channels.


Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Benzamides/pharmacology , Myocytes, Cardiac/physiology , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Receptors, Drug/antagonists & inhibitors , Action Potentials/drug effects , Adenosine Diphosphate/pharmacology , Animals , Guanidines/pharmacology , Iodoacetates/pharmacology , Male , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Pinacidil/pharmacology , Potassium Channels , Pyridines/pharmacology , Rats , Rats, Wistar , Sarcolemma/metabolism , Sodium Cyanide/pharmacology , Sulfonylurea Compounds/pharmacology , Sulfonylurea Receptors
6.
Curr Pharm Des ; 11(15): 1915-40, 2005.
Article in English | MEDLINE | ID: mdl-15974968

ABSTRACT

ATP-sensitive potassium (K(ATP)) channels link membrane excitability to metabolism. They are regulated by intracellular nucleotides and by other factors including membrane phospholipids, protein kinases and phosphatases. K(ATP) channels comprise octamers of four Kir6 pore-forming subunits associated with four sulphonylurea receptor subunits. The exact subunit composition differs between the tissues in which the channels are expressed, which include pancreas, cardiac, smooth and skeletal muscle and brain. K(ATP) channels are targets for antidiabetic sulphonylurea blockers, and for channel opening drugs that are used as antianginals and antihypertensives. This review focuses on non-pancreatic K(ATP) channels. In vascular smooth muscle, K(ATP) channels are extensively regulated by signalling pathways and cause vasodilation, contributing both to resting blood flow and vasodilator-induced increases in flow. Similarly, K(ATP) channel activation relaxes smooth muscle of the bladder, gastrointestinal tract and airways. In cardiac muscle, sarcolemmal K(ATP) channels open to protect cells under stress conditions such as ischaemia or exercise, and appear central to the protection induced by ischaemic preconditioning (IPC). Mitochondrial K(ATP) channels are also strongly implicated in IPC, but clarification of their exact role awaits information on their molecular structure. Skeletal muscle K(ATP) channels play roles in fatigue and recovery, K+ efflux, and glucose uptake, while neuronal channels may provide ischaemic protection and underlie the glucose-responsiveness of hypothalamic neurones. Current therapeutic considerations include the use of K(ATP) openers to protect cardiac muscle, attempts to develop openers selective for airway or bladder, and the question of whether block of extra-pancreatic K(ATP) channels may cause adverse cardiovascular side-effects of sulphonylureas.


Subject(s)
Adenosine Triphosphate/physiology , Potassium Channels/physiology , Animals , Humans , Muscle Relaxation/physiology , Muscle, Smooth, Vascular/physiology , Vasodilation/physiology
7.
J Mol Cell Cardiol ; 37(2): 579-91, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15276027

ABSTRACT

The protective effects of the PKC activator Phorbol 12-myristate 13-acetate (PMA) were investigated in electrically field stimulated (EFS) rat isolated ventricular myocytes following 7 min of metabolic inhibition induced by cyanide, iodoacetic acid and substrate removal, followed by reperfusion. PMA reduced reperfusion damage and increased functional recovery (response to EFS) following 10 min reperfusion from 20.0 +/- 10.7% of control myocytes to 90.0 +/- 7.2% following 5 min PMA pre-treatment (p<0.001). PMA significantly increased the time from the onset of MI before the myocytes failed to respond to EFS from 135 +/- 19s in control cells to 200 +/- 14s in PMA pre-treated cells (p<0.05). Additionally, there was an increase in the time to rigor with PMA pre-treated cells entering rigor 255 +/- 17s after MI compared to 174 +/-15s in control cell (p<0.05), indicating a delay in ATP depletion. During MI PMA pre-treated cells showed a significantly smaller increase in [Ca(2+)]i compared to control myocytes. Following reperfusion the majority of PMA pre-treated myocytes recovered calcium transients in response to EFS and diastolic Ca(2+) levels not significantly different to those seen prior to metabolic inhibition. Activation of PKC is thought to involve translocation to the particulate fraction. Our results demonstrate the presence of PKC-alpha, beta, gamma, delta, iota, lambda/zeta in rat ventricular myocytes, all of which translocate to the membrane in response to PMA.


Subject(s)
Myocardial Reperfusion Injury/prevention & control , Myocytes, Cardiac/enzymology , Protein Kinase C/metabolism , Animals , Calcium/metabolism , Electric Stimulation , Enzyme Activation/drug effects , Heart Ventricles/cytology , In Vitro Techniques , Isoenzymes/analysis , Isoenzymes/metabolism , Male , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/chemistry , Myocytes, Cardiac/drug effects , Protein Kinase C/analysis , Rats , Tetradecanoylphorbol Acetate/pharmacology
8.
J Physiol ; 557(Pt 3): 785-94, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15020694

ABSTRACT

C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K(+) channels (K(ATP)). To investigate the protective role of K(ATP) channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294-1358, the A-fragment, reduced sarcolemmal K(ATP) currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 +/- 1.22; and A-fragment 0.94 +/- 0.07 pA pF(-1), n= 6,6, P < 0.001). An inactive fragment (1358-1545, current density 6.30 +/- 0.85 pA pF(-1), n= 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca(2+) loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca(2+) and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca(2+) loading, was abolished by the A-fragment. Our data are consistent with a role for K(ATP) channels in causing action potential failure and reduced Ca(2+) loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K(ATP) channel, but we also discuss the possibility of mitochondrial effects.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Myocardial Ischemia/metabolism , Myocytes, Cardiac/metabolism , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/physiology , Potassium Channels/metabolism , Potassium Channels/physiology , Receptors, Drug/physiology , 2,4-Dinitrophenol/pharmacology , ATP-Binding Cassette Transporters/genetics , Action Potentials/drug effects , Animals , Antimetabolites/pharmacology , Calcium/metabolism , Calcium/physiology , Cells, Cultured , Electric Stimulation , Electrophysiology , In Vitro Techniques , KATP Channels , Male , Membrane Potentials/physiology , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Patch-Clamp Techniques , Peptide Fragments/genetics , Peptide Fragments/physiology , Potassium Channels/biosynthesis , Potassium Channels/genetics , Potassium Channels, Inwardly Rectifying/genetics , Rats , Rats, Wistar , Receptors, Drug/genetics , Sarcolemma/drug effects , Sarcolemma/metabolism , Sulfonylurea Receptors , Transfection
9.
J Membr Biol ; 192(2): 141-8, 2003 Mar 15.
Article in English | MEDLINE | ID: mdl-12682801

ABSTRACT

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are sensitive to both voltage and internal [Ca(2+)] and are found in many tissues. Their physiological roles range from causing relaxation of smooth muscle to regulating the frequency of action potential firing. There is considerable variation between different tissues in their Ca(2+)- and voltage-dependence. Much of this variation results from the association of the pore-forming alpha subunit (hSloalpha) with different beta subunits leading to altered channel properties. Since hSloalpha alone produces functional BK(Ca) channels, we have used a bicistronic expression method to ensure that both alpha and beta subunits are expressed, with the beta subunit being in excess. Using this method we have investigated the effect of four beta subunits (beta1 to beta4) on cloned BK(Ca) channels. The four beta subunits were individually cloned into a vector that had hSloalpha cDNA inserted downstream of an internal ribosome entry site. The constructs were transiently transfected into HEK293 cells together with a construct that expresses green fluorescent protein, as a marker for transfection. Fluorescent cells expressed BK(Ca) channels whose currents were recorded from inside-out or outside-out patches. The currents we measured using this expression system were similar to those expressed in Xenopus oocytes by Brenner et al. (Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. Cloning and functional expression of novel large-conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)


Subject(s)
Ion Channel Gating/physiology , Kidney/physiology , Membrane Potentials/physiology , Potassium Channels, Calcium-Activated/physiology , Cell Line , Cloning, Molecular , Dose-Response Relationship, Drug , Gene Expression Regulation/physiology , Humans , Ion Channel Gating/drug effects , Kidney/drug effects , Kidney/embryology , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits , Large-Conductance Calcium-Activated Potassium Channel beta Subunits , Large-Conductance Calcium-Activated Potassium Channels , Peptides/pharmacology , Potassium Channels, Calcium-Activated/chemistry , Potassium Channels, Calcium-Activated/genetics , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
11.
J Mol Cell Cardiol ; 34(5): 555-69, 2002 May.
Article in English | MEDLINE | ID: mdl-12056859

ABSTRACT

We have investigated the protective effects of pretreatment with the mitochondrial uncoupler 2,4-dinitrophenol on the cellular damage induced by metabolic inhibition (with cyanide and iodoacetic acid) and reperfusion in freshly isolated adult rat ventricular myocytes. Damage was assessed from changes in cell length and morphology measured using video microscopy. Intracellular Ca(2+), mitochondrial membrane potential, and NADH were measured using fura-2, tetramethylrhodamine ethyl ester and autofluorescence, respectively. During metabolic inhibition myocytes developed rigor, and on reperfusion 73.6+/-8.1% hypercontracted and 10.8+/-6.7% recovered contractile function in response to electrical stimulation. Intracellular Ca(2+) increased substantially, indicated by a rise in the fura-2 ratio (340/380 nm) on reperfusion from 0.86+/-0.04 to 1.93+/-0.18. Myocytes pretreated with substrate-free Tyrode containing 50 microm dinitrophenol showed reduced reperfusion injury: 29.0+/-7.4% of cells hypercontracted and 65.3+/-7.3% recovered contractile function (P<0.001 vs control). The fura-2 ratio on reperfusion was also lower at 1.01+/-0.08. Fluorescence measurements showed that dinitrophenol caused mitochondrial depolarisation, and decreased NADH. The presence of the substrates glucose and pyruvate reduced these effects, and abolished the protection against damage by metabolic inhibition and reperfusion. However protection was unaffected by block of ATP-sensitive potassium channels. Thus the protective effects of pretreatment with dinitrophenol may result from a reduction in NADH in response to mitochondrial depolarisation.


Subject(s)
Dinitrophenols/pharmacology , Muscle Fibers, Skeletal/metabolism , Myocardial Reperfusion Injury/prevention & control , Myocardium/metabolism , Uncoupling Agents/pharmacology , Animals , Cyanides/toxicity , Iodoacetic Acid/toxicity , Ischemic Preconditioning, Myocardial , Male , Mitochondria/metabolism , Muscle Fibers, Skeletal/pathology , Myocardial Contraction/drug effects , Myocardial Reperfusion Injury/chemically induced , Myocardium/pathology , NAD/metabolism , Rats , Rats, Wistar
12.
Br J Pharmacol ; 136(5): 746-52, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12086984

ABSTRACT

1. We have investigated the effects of the sulphonylurea, glimepiride, currently used to treat type 2 diabetes, on ATP-sensitive K(+) (K(ATP)) currents of rat cardiac myocytes and on their cloned constituents Kir6.2 and SUR2A expressed in HEK 293 cells. 2. Glimepiride blocked pinacidil-activated whole-cell K(ATP) currents of cardiac myocytes with an IC(50) of 6.8 nM, comparable to the potency of glibenclamide in these cells. Glimepiride blocked K(ATP) channels formed by co-expression of Kir6.2/SUR2A subunits in HEK 293 cells in outside-out excised patches with a similar IC(50) of 6.2 nM. 3. Glimepiride was much less effective at blocking K(ATP) currents activated by either metabolic inhibition (MI) with CN(-) and iodoacetate or by the K(ATP) channel opener diazoxide in the presence of inhibitors of F(0)/F(1)-ATPase (oligomycin) and creatine kinase (DNFB). Thus 10 microM glimepiride blocked pinacidil-activated currents by >99%, MI-activated currents by 70% and diazoxide-activated currents by 82%. 4. In inside-out patches from HEK 293 cells expressing the cloned K(ATP) channel subunits Kir6.2/SUR2A, increasing the concentration of ADP (1 - 100 microM), in the presence of 100 nM glimepiride, lead to significant increases in Kir6.2/SUR2A channel activity. However, over the range tested, ADP did not affect cloned K(ATP) channel activity in the presence of 100 nM glibenclamide. These results are consistent with the suggestion that ADP reduces glimepiride block of K(ATP) channels. 5. Our results show that glimepiride is a potent blocker of native cardiac K(ATP) channels activated by pinacidil and blocks cloned Kir6.2/SUR2A channels activated by ATP depletion with similar potency. However, glimepiride is much less effective when K(ATP) channels are activated by MI and this may reflect a reduction in glimepiride block by increased intracellular ADP.


Subject(s)
Myocardium/metabolism , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Sarcolemma/drug effects , Sulfonylurea Compounds/pharmacology , Animals , Humans , Male , Neural Inhibition/drug effects , Neural Inhibition/physiology , Potassium Channels/metabolism , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/metabolism , Rats , Rats, Wistar , Sarcolemma/metabolism
13.
Am J Physiol Heart Circ Physiol ; 281(6): H2480-9, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11709415

ABSTRACT

The vasoconstrictor angiotensin II (ANG II) inhibits several types of K(+) channels. We examined the inhibitory mechanism of ANG II on voltage-gated K(+) (K(V)) currents (I(K(V))) recorded from isolated rat arterial smooth muscle using patch-clamp techniques. Application of 100 nM ANG II accelerated the activation of I(K(V)) but also caused inactivation. These effects were abolished by the AT(1) receptor antagonist losartan. The protein kinase A (PKA) inhibitor Rp-cyclic 3',5'-hydrogen phosphothioate adenosine (100 microM) and an analog of diacylglycerol, 1,2-dioctanyoyl-rac-glycerol (2 microM), caused a significant reduction of I(K(V)). Furthermore, the combination of 5 microM PKA inhibitor peptide 5-24 (PKA-IP) and 100 microM protein kinase C (PKC) inhibitor peptide 19-27 (PKC-IP) prevented the inhibition by ANG II, although neither alone was effective. The ANG II effect seen in the presence of PKA-IP remained during addition of the Ca(2+)-dependent PKC inhibitor Gö6976 (1 microM) but was abolished in the presence of 40 microM PKC-epsilon translocation inhibitor peptide. These results demonstrate that ANG II inhibits K(V) channels through both activation of PKC-epsilon and inhibition of PKA.


Subject(s)
Angiotensin II/pharmacology , Cyclic AMP/analogs & derivatives , Muscle, Smooth, Vascular/enzymology , Potassium Channels, Voltage-Gated/metabolism , Vasoconstrictor Agents/pharmacology , Animals , Antihypertensive Agents/pharmacology , Cyclic AMP/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Diglycerides/pharmacology , Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Kinetics , Losartan/pharmacology , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mesenteric Arteries/cytology , Mesenteric Arteries/metabolism , Muscle, Smooth, Vascular/cytology , Patch-Clamp Techniques , Peptides/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase C-epsilon , Rats , Rats, Wistar , Thionucleotides/pharmacology
14.
J Am Coll Cardiol ; 37(3): 711-8, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11693741

ABSTRACT

OBJECTIVES: We investigated the effects of ischemic preconditioning (PC) on diabetic and failing human myocardium and the role of mitochondrial KATP channels on the response in these diseased tissues. BACKGROUND: There is conflicting evidence to suggest that PC is a healthy heart phenomenon. METHODS: Right atrial appendages were obtained from seven different groups of patients: nondiabetics, diet-controlled diabetics, noninsulin-dependent diabetics (NIDD) receiving KATP channel blockers, insulin-dependent diabetics (IDD), and patients with left ventricular ejection fraction (LVEF) >50%, LVEF between 30% and 50% and LVEF <30%. After stabilization, the muscle slices were randomized into five experimental groups (n = 6/group): 1) aerobic control-incubated in oxygenated buffer for 210 min, 2) ischemia alone-90 min ischemia followed by 120 min reoxygenation, 3) preconditioning by 5 min ischemia/5 min reoxygenation before 90 min ischemia/120 min reoxygenation, 4) diazoxide (Mito KATP opener, 0.1 mm)-for 10 min before the 90 min ischemia/120 min reoxygenation and 5) glibenclamide (10 microm)-10 min exposure prior to PC (only in the diabetic patient groups). Creatine kinase leakage into the medium (CK, U/g wet wt) and MTT reduction (OD/mg wet wt), an index of cell viability, were assessed at the end of the experiment. RESULTS: Ischemia caused similar injury in both normal and diseased tissue. Preconditioning prevented the effects of ischemia in all groups except NIDD, IDD and poor cardiac function (<30%). In the diazoxide-treated groups, protection was mimicked in all groups except the NIDD and IDD groups. Interestingly, glybenclamide abolished protection in nondiabetic and diet-controlled NIDD groups and did not affect NIDD groups receiving KATP channel blockers or IDD groups. CONCLUSIONS: These results show that failure to precondition the diabetic heart is due to dysfunction of the mitochondrial KATP channels and that the mechanism of failure in the diabetic heart lies in elements of the signal transduction pathway different from the mitochondrial KATP channels.


Subject(s)
Adenosine Triphosphate/physiology , Diabetic Angiopathies/physiopathology , Ischemic Preconditioning, Myocardial , Mitochondria, Heart/physiology , Potassium Channels/metabolism , Aged , Female , Humans , In Vitro Techniques , Male , Middle Aged , Signal Transduction/physiology
15.
J Physiol ; 536(Pt 2): 421-7, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11600677

ABSTRACT

1. We have investigated the possible role of A-kinase anchoring proteins (AKAPs) in protein kinase A (PKA) signalling to ATP-sensitive K+ (K(ATP)) channels of rat isolated mesenteric arterial smooth muscle cells using whole-cell patch clamp and peptides that inhibit PKA-AKAP binding. 2. Intracellular Ht31 peptide (20 microM), which inhibits the PKA-AKAP interaction, blocked K(ATP) current activation by either dibutyryl cAMP or calcitonin gene-related peptide. Ht31-proline (20 microM), which does not inhibit PKA binding to AKAP, did not block K(ATP) current activation. 3. Ht31 reduced K(ATP) current activated by pinacidil and also prevented its inhibition by Rp-cAMPS, effects consistent with Ht31 blocking steady-state K(ATP) channel activation by PKA. However, Ht31 did not prevent K(ATP) current activation by the catalytic subunit of PKA. 4. An antibody to the RII subunit of PKA showed localization of PKA near to the cell membrane. Our results provide evidence that both steady-state and receptor-driven activation of K(ATP) channels by PKA involve the localization of PKA by an AKAP.


Subject(s)
Carrier Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Muscle, Smooth, Vascular/enzymology , Potassium Channels/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Bucladesine/pharmacology , Calcitonin Gene-Related Peptide/pharmacology , Cell Membrane/enzymology , Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Male , Mesenteric Arteries/cytology , Molecular Sequence Data , Muscle, Smooth, Vascular/cytology , Patch-Clamp Techniques , Peptide Fragments/pharmacology , Rats , Rats, Wistar
16.
Br J Pharmacol ; 134(3): 535-42, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11588107

ABSTRACT

1. The K(ATP) channel opener diazoxide has been proposed to protect cardiac muscle against ischaemia by opening mitochondrial K(ATP) channels to depolarize the mitochondrial membrane potential, DeltaPsi(m). We have used the fluorescent dye TMRE to measure DeltaPsi(m) in adult rat freshly isolated cardiac myocytes exposed to diazoxide and metabolic inhibition. 2. Diazoxide, at concentrations that are highly cardioprotective (100 or 200 microM), caused no detectable increase in TMRE fluorescence (n=27 cells). However, subsequent application of the protonophore FCCP, which should collapse DeltaPsi(m), led to large increases in TMRE fluorescence (>300%). 3. Metabolic inhibition (MI: 2 mM NaCN+1 mM iodoacetic acid (IAA) led to an immediate partial depolarization of DeltaPsi(m), followed after a few minutes delay by complete depolarization which was correlated with rigor contracture. Removal of metabolic inhibition led to abrupt mitochondrial repolarization followed in many cells by hypercontracture, indicated by cell rounding and loss of striated appearance. 4. Prior application of diazoxide (100 microM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7+/-4.7% to 24.2+/-1.8% (P< 0.0001). 5-hydroxydeanoate (100 microM) reduced the protection of diazoxide (46.8+/-2.7% cells hypercontracted, P< 0.0001 vs diazoxide alone). 5. Diazoxide caused no detectable change in flavoprotein autofluorescence (n=26 cells). 6. Our results suggest that mitochondrial depolarization and flavoprotein oxidation are not inevitable consequences of diazoxide application in intact cardiac myocytes, and that they are also not essential components of the mechanism by which it causes protection.


Subject(s)
Diazoxide/pharmacology , Flavoproteins/metabolism , Mitochondria, Heart/drug effects , Myocardium/cytology , Myocardium/metabolism , Potassium Channels/metabolism , Vasodilator Agents/pharmacology , Animals , Cell Separation , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mitochondria, Heart/metabolism , Oxidation-Reduction , Rats , Rats, Wistar
17.
Diabetologia ; 44(8): 1019-25, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11484080

ABSTRACT

AIMS/HYPOTHESIS: Sulphonylureas stimulate insulin secretion by closing ATP-sensitive potassium (KATP) channels in the pancreatic beta-cell membrane. KATP channels are also found in other tissues, including heart and smooth muscle, where they link cellular metabolism to electrical activity. The sulphonylurea gliclazide blocks recombinant beta-cell KATP channels (Kir6.2/SUR1) but not heart (Kir6.2/SUR2A) or smooth muscle (Kir6.2/SUR2B) KATP channels with high potency. In this study, we examined the specificity of gliclazide for the native (as opposed to recombinant) KATP channels in beta cells, heart and smooth muscle. METHODS: The action of the drug was studied by whole-cell current recordings of native KATP channels in isolated pancreatic beta-cells and myocytes from heart and smooth muscle. RESULTS: Gliclazide blocked whole-cell beta-cell KATP currents with an IC50 of 184 +/- 30 nmol/l (n = 6-10) but was much less effective in cardiac and smooth muscle (IC50s of 19.5 +/- 5.4 micromol/l (n = 6-12) and 37.9 +/- 1.0 micromol/l (n = 5-10), respectively). In all three tissues, the action of the drug on whole-cell KATP currents was rapidly reversible. In inside-out patches on beta-cells, gliclazide (1 micromol/l) produced a maximum of 66 +/- 13 % inhibition (n = 5), compared with more than 98 % block in the whole-cell configuration. CONCLUSION/INTERPRETATION: Gliclazide is a high-potency sulphonylurea which shows specificity for the pancreatic beta-cell KATP channel over heart and smooth muscle. In this respect, it differs from glibenclamide. The difference in the maximal block observed in the excised patch and whole-cell recordings from beta-cells, may be due to the absence of intracellular Mg-nucleotides in the excised patch experiments.


Subject(s)
Gliclazide/pharmacology , Heart/drug effects , Islets of Langerhans/drug effects , Muscle, Smooth, Vascular/drug effects , Potassium Channel Blockers , Potassium Channels, Inwardly Rectifying , Animals , Electric Conductivity , Heart/physiology , Islets of Langerhans/physiology , Male , Mice , Muscle, Smooth, Vascular/physiology , Potassium Channels/physiology , Rats , Rats, Wistar
18.
Circulation ; 103(25): 3111-6, 2001 Jun 26.
Article in English | MEDLINE | ID: mdl-11425777

ABSTRACT

BACKGROUND: The sulfonylurea glibenclamide (Glib) abolishes the cardioprotective effect of ischemic preconditioning (IP), presumably by inhibiting mitochondrial K(ATP) channel opening in myocytes. Glimepiride (Glim) is a new sulfonylurea reported to affect nonpancreatic K(ATP) channels less than does Glib. We examined the effects of Glim on IP and on the protection afforded by diazoxide (Diaz), an opener of mitochondrial K(ATP) channels. METHODS AND RESULTS: Rat hearts were Langendorff-perfused, subjected to 35 minutes of regional ischemia and 120 minutes of reperfusion, and assigned to 1 of the following treatment groups: (1) control; (2) IP of 2x 5 minutes each of global ischemia before lethal ischemia; or pretreatment with (3) 30 micromol/L Diaz, (4) 10 micromol/L Glim, (5) 10 micromol/L Glib, (6) IP+Glim, (7) IP+Glib, (8) Diaz+Glim, or (9) Diaz+Glib. IP limited infarct size (18.5+/-1% vs 43.7+/-3% in control, P<0.01) as did Diaz (22.2+/-4.7%, P<0.01). The protective actions of IP or Diaz were not abolished by Glim (18.5+/-3% in IP+Glim, 22.3+/-3% in Diaz+Glim; P<0.01 vs control). However, Glib abolished the infarct-limiting effects of IP and Diaz. Patch-clamp studies in isolated rat ventricular myocytes confirmed that both Glim and Glib (each at 1 micromol/L) blocked sarcolemmal K(ATP) currents. However, in isolated cardiac mitochondria, Glim (10 micromol/L) failed to block the effects of K(ATP) opening by GTP, in contrast to the blockade caused by Glib. CONCLUSIONS: Although it blocks sarcolemmal currents in rat cardiac myocytes, Glim does not block the beneficial effects of mitochondrial K(ATP) channel opening in the isolated rat heart. These data may have significant implications for the treatment of type 2 diabetes in patients with ongoing ischemic heart disease.


Subject(s)
Diazoxide/pharmacology , Hypoglycemic Agents/pharmacology , Ischemic Preconditioning, Myocardial , Myocardial Ischemia/prevention & control , Sulfonylurea Compounds/pharmacology , Animals , Glyburide/pharmacology , Guanosine Triphosphate/pharmacology , Heart/drug effects , Heart/physiopathology , Heart Ventricles/cytology , Heart Ventricles/drug effects , Hemodynamics/drug effects , In Vitro Techniques , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Male , Membrane Potentials/drug effects , Membrane Proteins/drug effects , Mitochondria, Heart/drug effects , Mitochondria, Heart/physiology , Myocardial Infarction/etiology , Myocardial Infarction/pathology , Myocardial Infarction/prevention & control , Myocardial Ischemia/complications , Patch-Clamp Techniques , Potassium Channels , Rats , Rats, Sprague-Dawley , Ventricular Function
19.
FEBS Lett ; 492(1-2): 146-50, 2001 Mar 09.
Article in English | MEDLINE | ID: mdl-11248253

ABSTRACT

Selective protein kinase C (PKC) activators and inhibitors were used to investigate the involvement of specific PKC isoforms in the modulation of voltage-sensitive Ca(2+) channels (VSCCs) in bovine adrenal chromaffin cells. Exposure to the phorbol ester phorbol-12,13-dibutyrate (PDBu) inhibited the Ca(2+) currents elicited by depolarizing voltage steps. This inhibition was occluded by the PKC-specific inhibitor Ro 31-8220 but remained unaffected by Gö 6976, a selective inhibitor of conventional PKC isoforms. PDBu treatment caused the translocation of PKC-alpha and -epsilon isoforms from cytosol to membranes. PKC-iota and -zeta showed no signs of translocation. It is concluded that VSCCs are specifically inhibited by the activation of PKC-epsilon in chromaffin cells. This may be relevant to the action of phospholipase-linked receptors involved in the control of Ca(2+) influx, both in catecholaminergic cells and other cell types.


Subject(s)
Calcium Channels/metabolism , Chromaffin Cells/enzymology , Protein Kinase C/metabolism , Adrenal Glands/enzymology , Animals , Calcium Channels/drug effects , Cattle , Chromaffin Cells/drug effects , Chromaffin Cells/metabolism , In Vitro Techniques , Phorbol Esters/pharmacology , Protein Isoforms/drug effects , Protein Isoforms/metabolism
20.
J Physiol ; 530(Pt 2): 193-205, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11208968

ABSTRACT

We used whole-cell patch clamp to investigate steady-state activation of ATP-sensitive K+ channels (KATP) of rat arterial smooth muscle by protein kinase A (PKA) and the pathway by which angiotensin II (Ang II) inhibits these channels. Rp-cAMPS, an inhibitor of PKA, did not affect KATP currents activated by pinacidil when the intracellular solution contained 0.1 mM ATP. However, when ATP was increased to 1.0 mM, inhibition of PKA reduced KATP current, while the phosphatase inhibitor calyculin A caused a small increase in current. Ang II (100 nM) inhibited KATP current activated by the K+ channel opener pinacidil. The degree of inhibition was greater with 1.0 mM than with 0.1 mM intracellular ATP. The effect of Ang II was abolished by the AT1 receptor antagonist losartan. The inhibition of KATP currents by Ang II was abolished by a combination of PKA inhibitor peptide 5-24 (5 microM) and PKC inhibitor peptide 19-27 (100 microM), while either alone caused only partial block of the effect. In the presence of PKA inhibitor peptide, the inhibitory effect of Ang II was unaffected by the PKC inhibitor Go 6976, which is selective for Ca2+-dependent isoforms of PKC, but was abolished by a selective peptide inhibitor of the translocation of the epsilon isoform of PKC. Our results indicate that KATP channels are activated by steady-state phosphorylation by PKA at normal intracellular ATP levels, and that Ang II inhibits the channels both through activation of PKCepsilon and inhibition of PKA.


Subject(s)
Adenosine Triphosphate/physiology , Angiotensin II/pharmacology , Arteries/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Isoenzymes/metabolism , Potassium Channel Blockers , Protein Kinase C/metabolism , Adenosine Triphosphate/pharmacology , Angiotensin II/antagonists & inhibitors , Angiotensin II Type 1 Receptor Blockers , Angiotensin II Type 2 Receptor Blockers , Angiotensin Receptor Antagonists , Animals , Cyclic AMP-Dependent Protein Kinases/physiology , Drug Combinations , Electric Conductivity , Enzyme Activation , Enzyme Inhibitors/pharmacology , Homeostasis , Isoenzymes/physiology , Male , Patch-Clamp Techniques , Potassium Channels/physiology , Protein Kinase C/physiology , Protein Kinase C-epsilon , Rats , Rats, Wistar , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Receptors, Angiotensin/physiology
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