Kir6.2 limits Ca(2+) overload and mitochondrial oscillations of ventricular myocytes in response to metabolic stress.

ATP-sensitive K(+) (KATP) channels are abundant membrane proteins in cardiac myocytes that are directly gated by intracellular ATP and form a signaling complex with metabolic enzymes, such as creatine kinase. KATP channels are known to be essential for adaption to cardiac stress, such as ischemia; however, how all the molecular components of the stress response interact is not fully understood. We examined the effects of decreasing the KATP current density on Ca(2+) and mitochondrial homeostasis and ischemic preconditioning. Acute knockdown of the pore-forming subunit, Kir6.2, was achieved using adenoviral delivery of short hairpin RNA targeted to Kir6.2. The acute nature of the knockdown of Kir6.2 accurately shows the effects of Kir6.2 depletion without any compensatory effects that may arise in transgenic studies. We also investigated the effect of reducing the KATP current while maintaining KATP channel protein in the sarcolemmal membrane using a nonconducting Kir6.2 construct. Only 50% KATP current remained after Kir6.2 knockdown, yet there were profound effects on myocyte responses to metabolic stress. Kir6.2 was essential for cardiac myocyte Ca(2+) homeostasis under both baseline conditions before any metabolic stress and after metabolic stress. Expression of nonconducting Kir6.2 also resulted in increased Ca(2+) overload, showing the importance of K(+) conductance in the protective response. Both ischemic preconditioning and protection during ischemia were lost when Kir6.2 was knocked down. KATP current density was also important for the mitochondrial membrane potential at rest and prevented mitochondrial membrane potential oscillations during oxidative stress. KATP channel density is important for adaption to metabolic stress.

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle.

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.

Phenylephrine preconditioning involves modulation of cardiac sarcolemmal K(ATP) current by PKC delta, AMPK and p38 MAPK.

Preconditioning of hearts with the α(1)-adrenoceptor agonist phenylephrine decreases infarct size and increases the functional recovery of the heart following ischaemia-reperfusion. However, the cellular mechanisms responsible for this protection are not known. We investigated the role of protein kinase C ε and δ (PKCε and PKCδ), AMP-activated protein kinase (AMPK), p38 MAPK (p38) and sarcolemmal ATP-sensitive potassium (sarcK(ATP)) channels in phenylephrine preconditioning using isolated rat ventricular myocytes. Preconditioning of ventricular myocytes with phenylephrine increased the recovery of contractile activity following metabolic inhibition and re-energisation from 30.1±1.9% to 66.5±5.2% (P<0.01) and increased the peak sarcK(ATP) current activated during metabolic inhibition from 32.1±1.8 pA/pF to 46.0±5.0 pA/pF (P<0.05), which was required for protection. Phenylephrine preconditioning resulted in a sustained activation of PKCε and PKCδ, and transient activation of AMPK, which was dependent upon activation of PKCδ but not PKCε. P38 was also activated by phenylephrine preconditioning and this was blocked by inhibitors of PKCε, PKCδ or AMPK. Inhibition of PKCδ, AMPK or p38 was sufficient to prevent the increase in current, suggesting that these kinases are involved in modulation of sarcK(ATP) channel current by phenylephrine preconditioning. However, whilst inhibition of AMPK and p38 prevented the protection from phenylephrine preconditioning, PKCδ inhibition paradoxically had no effect. The increase in sarcK(ATP) current induced by phenylephrine preconditioning requires PKCδ, AMPK and p38 and may contribute to the observed improvement in contractile recovery.

Principal role of adenylyl cyclase 6 in K⁺ channel regulation and vasodilator signalling in vascular smooth muscle cells.

AIMS: Membrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e.g. the ATP-sensitive potassium channel (K(ATP))] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential. METHODS AND RESULTS: AC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for ∼60% of the total response to ß-adrenoceptor (ß-AR) stimulation. AC3 played a minor role in ß-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in ß-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial ß-AR/PKA-dependent enhancement of K(ATP) current. K(ATP) current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon ß-AR stimulation. CONCLUSION: AC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure.

Modulation of the nitric oxide metabolism overcomes the unresponsiveness of the diabetic human myocardium to protection against ischemic injury.

BACKGROUND: We have demonstrated that diabetic human myocardium cannot be protected by ischemic preconditioning (IP) and identified a dysfunction of the mitochondria as the cause of the defect. Here we have investigated whether modulation of the nitric oxide (NO) metabolism can overcome the unresponsiveness of the diabetic myocardium to cardioprotection. METHODS: Myocardial slices (30-40 mg) obtained from the right atrial appendage of patients with diabetes undergoing elective cardiac surgery were randomized to the following protocol (n=6/group): NO donor SNAP (100 µM), nonselective nitric oxide synthase (NOS) inhibitor L-NAME (100 µM), and selective neuronal NOS (nNOS) inhibitor TRIM (100 µM) for 20 min prior to 90 min ischemia followed by 120 min reoxygenation (37°C). Some preparations were subjected to ischemic/reoxygenation alone or to IP (5 min ischemia/5 min reoxygenation) to act as control. Tissue injury was assessed by creatine kinase (CK) released (IU/mg wet wt), and cell necrosis and apoptosis by propidium iodide and TUNEL (% of aerobic control). RESULTS: IP did not decrease CK release, cell necrosis or apoptosis in diabetic myocardium. However, NO donor SNAP, the nonspecific NOS inhibitor L-NAME, and the specific nNOS inhibitor TRIM significantly reduced CK leakage, cell necrosis, and apoptosis in diabetic myocardium. CONCLUSIONS: These results demonstrate that both the provision of exogenous NO and the suppression of endogenous NO production result in potent protection of diabetic human myocardium overcoming the unresponsiveness of these tissues to cardioprotective therapies.

G protein-coupled receptor kinase 2 and arrestin2 regulate arterial smooth muscle P2Y-purinoceptor signalling.

AIMS: prolonged P2Y-receptor signalling can cause vasoconstriction leading to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs) and arrestin proteins, preventing prolonged or inappropriate signalling. This study investigates whether GRKs and arrestins regulate uridine 5'-triphosphate (UTP)-stimulated contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). METHODS AND RESULTS: mesenteric arteries contracted in response to UTP challenge: When an EC(50) UTP concentration (30 µM, 5 min) was added 5 min before (R(1)) and after (R(2)) the addition of a maximal UTP concentration (R(max): 100 µM, 5 min), R(2) responses were decreased relative to R(1), indicating desensitization. UTP-induced P2Y-receptor desensitization of phospholipase C signalling was studied in isolated MSMCs transfected with an inositol 1,4,5-trisphosphate biosensor and/or loaded with Ca(2+)-sensitive dyes. A similar protocol (R(1)/R(2) = 10 µM; R(max) = 100 µM, applied for 30 s) revealed markedly reduced R(2) when compared with R(1) responses. MSMCs were transfected with dominant-negative GRKs or siRNAs targeting specific GRK/arrestins to probe their respective roles in P2Y-receptor desensitization. GRK2 inhibition, but not GRK3, GRK5, or GRK6, attenuated P2Y-receptor desensitization. siRNA-mediated knockdown of arrestin2 attenuated UTP-stimulated P2Y-receptor desensitization, whereas arrestin3 depletion did not. Specific siRNA knockdown of the P2Y(2)-receptor almost completely abolished UTP-stimulated IP(3)/Ca(2+) signalling, strongly suggesting that our study is specifically characterizing this purinoceptor subtype. CONCLUSION: these new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y(2)-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.

Dual role of nNOS in ischemic injury and preconditioning.

BACKGROUND: Nitric oxide (NO) is cardioprotective and a mediator of ischemic preconditioning (IP). Endothelial nitric oxide synthase (eNOS) is protective against myocardial ischemic injury and a component of IP but the role and location of neuronal nitric oxide synthase (nNOS) remains unclear. Therefore, the aims of these studies were to: (i) investigate the role of nNOS in ischemia/reoxygenation-induced injury and IP, (ii) determine whether its effect is species-dependent, and (iii) elucidate the relationship of nNOS with mitoKATP channels and p38MAPK, two key components of IP transduction pathway. RESULTS: Ventricular myocardial slices from rats and wild and nNOS knockout mice, and right atrial myocardial slices from human were subjected to 90 min ischemia and 120 min reoxygenation (37 degrees C). Specimens were randomized to receive various treatments (n = 6/group). Both the provision of exogenous NO and the inhibition of endogenous NO production significantly reduced tissue injury (creatine kinase release, cell necrosis and apoptosis), an effect that was species-independent. The cardioprotection seen with nNOS inhibition was as potent as that of IP, however, in nNOS knockout mice the cardioprotective effect of non-selective NOS (L-NAME) and selective nNOS inhibition and also that of IP was blocked while the benefit of exogenous NO remained intact. Additional studies revealed that the cardioprotection afforded by exogenous NO and by inhibition of nNOS were unaffected by the mitoKATP channel blocker 5-HD, although it was abrogated by p38MAPK blocker SB203580. CONCLUSIONS: nNOS plays a dual role in ischemia/reoxygenation in that its presence is necessary to afford cardioprotection by IP and its inhibition reduces myocardial ischemic injury. The role of nNOS is species-independent and exerted downstream of the mitoKATP channels and upstream of p38MAPK.

Endothelin signalling in arterial smooth muscle is tightly regulated by G protein-coupled receptor kinase 2.

AIMS: Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). METHODS AND RESULTS: ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCdelta1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET(A)R) antagonist, BQ123. To characterize ET(A)R desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET(A)R responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET(A)R desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive (D110A,K220R)GRK2, (D110A,K220R)GRK3, (K215R)GRK5, or (K215R)GRK6 constructs. (D110A,K220R)GRK2 expression significantly attenuated ET(A)R desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET(A)R desensitization. Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane. CONCLUSION: These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

Endothelin-I and angiotensin II inhibit arterial voltage-gated K+ channels through different protein kinase C isoenzymes.

AIMS: Voltage-gated K+ (Kv) channels of arterial smooth muscle (ASM) modulate arterial tone and are inhibited by vasoconstrictors through protein kinase C (PKC). We aimed to determine whether endothelin-1 (ET-1) and angiotensin II (AngII), which cause similar inhibition of Kv, use the same signalling pathway and PKC isoenzyme to exert their effects on Kv and to compare the involvement of PKC isoenzymes in contractile responses to these agents. METHODS AND RESULTS: Kv currents recorded using the patch clamp technique with freshly isolated rat mesenteric ASM cells were inhibited by ET-1 or AngII. Inclusion of a PKCepsilon inhibitor peptide in the intracellular solution substantially reduced inhibition by AngII, but did not affect that by ET-1. Kv inhibition by ET-1 was reduced by the conventional PKC inhibitor Gö 6976 but not by the PKCbeta inhibitor LY333531. Selective peptide inhibitors of PKCalpha and PKCepsilon were linked to a Tat carrier peptide to make them membrane permeable and used to show that inhibition of PKCalpha prevented ET-1 inhibition of Kv current, but did not affect that by AngII. In contrast, inhibition of PKCepsilon prevented Kv inhibition by AngII but not by ET-1. The Tat-linked inhibitor peptides were also used to investigate the involvement of PKCalpha and PKCepsilon in the contractile responses of mesenteric arterial rings, showing that ET-1 contractions were substantially reduced by inhibition of PKCalpha, but unaffected by inhibition of PKCepsilon. AngII contractions were unaffected by inhibition of PKCalpha but substantially reduced by inhibition of PKCepsilon. CONCLUSION: ET-1 inhibits Kv channels of mesenteric ASM through activation of PKCalpha, while AngII does so through PKCepsilon. This implies that ET-1 and AngII target Kv channels of ASM through different pathways of PKC-interacting proteins, so each vasoconstrictor enables its distinct PKC isoenzyme to interact functionally with the Kv channel.

Visualizing the temporal effects of vasoconstrictors on PKC translocation and Ca2+ signaling in single resistance arterial smooth muscle cells.

Arterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of enhanced green fluorescence protein (eGFP)-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca(2+) signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes alpha, delta, and epsilon; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCalpha mirrored the rise in [Ca(2+)](i), while that of eGFP-PKCdelta or -epsilon occurred more slowly. Endothelin-induced translocation of eGFP-PKCepsilon was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca(2+)](i) increases using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCalpha translocation, while eGFP-PKCdelta translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment and demonstrate the varied kinetics and complex interplay between Ca(2+) and PKC responses to different vasoconstrictors in ASM.

Mitochondrial dysfunction as the cause of the failure to precondition the diabetic human myocardium.

OBJECTIVES: We have shown previously that human diabetic myocardium cannot be preconditioned. Here, we have investigated the basis of this cardioprotective deficit. METHODS: Right atrial sections from four patient groups-non-diabetic, insulin-dependent diabetes mellitus (IDDM), non-insulin-dependent diabetes mellitus (NIDDM) receiving glibenclamide, and NIDDM receiving metformin-were subjected to one of the following protocols: aerobic control, simulated ischemia/reoxygenation, ischemic preconditioning before ischemia, and pharmacological preconditioning with alpha 1 agonist phenylephrine, adenosine, the mito-K(ATP) channel opener diazoxide, the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA), or the p38 mitogen-activated protein kinase (p38MAPK) activator anisomycin. Cellular damage was assessed using creatine kinase leakage and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. In mitochondrial preparations from non-diabetic and diabetic myocardium, mitochondrial membrane potential (Psi(m)) was assessed using JC-1 dye, and production of reactive oxygen species was determined. RESULTS: Preconditioning with ischemia, phenylephrine, adenosine, or diazoxide failed to protect diabetic myocardium. However, activation of PKC or p38MAPK was still protective. In isolated non-diabetic mitochondria, diazoxide partially depolarized Psi(m), an effect not seen in diabetic mitochondria. Furthermore, diazoxide increased superoxide production in non-diabetic but not in diabetic mitochondria. CONCLUSIONS: Our results show that the cardioprotective deficit in diabetic myocardium arises upstream of PKC and p38MAPK. We suggest that mitochondrial dysfunction in diabetic myocardium, possibly dysfunctional mito-K(ATP) channels, leads to impaired depolarization and superoxide production, and that this causes the inability to respond to preconditioning.

The effect of gliclazide and glibenclamide on preconditioning of the human myocardium.

The cardioprotection of ischaemic preconditioning may be abolished in diabetic patients especially when some oral hypoglycaemics are used. The dose-response effect of gliclazide and glibenclamide on ischaemic preconditioning and the action of glibenclamide on signal transduction in human myocardium were investigated using right atrial appendages from cardiac surgery patients. Glibenclamide (0.1, 1, 3 and 10 microM) and gliclazide (1, 10, 30 and 100 microM) were added for 10 min prior to ischaemic preconditioning. The cardioprotection was abolished by glibenclamide at all concentrations and by gliclazide at supratherapeutic concentrations of 30 and 100 microM. Glibenclamide abolished the protective effect of mitoK(ATP) channel opening but not that of protein kinase C (PKC) or p38 mitogen activated protein kinase (p38MAPK) activation. In conclusion, glibenclamide and gliclazide differential effects may be a result of differential sensitivities. Glibenclamide does not block protection conferred by either PKC or p38MAPK activation. These findings may have clinical implications in ischaemic heart disease.

Role of mitochondrial re-energization and Ca2+ influx in reperfusion injury of metabolically inhibited cardiac myocytes.

OBJECTIVE: We used isolated myocytes to investigate the role of mitochondrial re-energization and Ca2+ influx during reperfusion on hypercontracture, loss of Ca2+ homeostasis and contractile function. METHODS: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion injury was assessed from hypercontracture, loss of Ca2+ homeostasis ([Ca2+]i measured with fura-2) and failure of contraction in response to electrical stimulation. Mitochondrial membrane potential was followed using the potentiometric dye tetramethylrhodamine ethyl ester. RESULTS: Metabolic inhibition led to contractile failure and rigor accompanied by a sustained increase in [Ca2+]i. Reperfusion after 10 min metabolic inhibition led to an abrupt repolarization of the mitochondrial membrane potential (after 25.5+/-1.2 s), a transient fall in [Ca(2+]i followed by an abrupt hypercontracture (37.1+/-1.8 s) in 84% of myocytes. Ca2+ homeostasis (diastolic [Ca2+]i < 250 nM) recovered in only 23.3+/-5.1% of cells and contractions recovered in 15.3+/-2.2%. Oligomycin abolished the hypercontracture on reperfusion, but mitochondrial repolarization was unaffected. Preventing Ca2+ influx during reperfusion with [Ca2+]i-free Tyrode or with an inhibitor of Na(+)/Ca2+ exchange did not prevent the hypercontracture, but increased the percentage of cells recovering Ca2+ homeostasis and contractile function. The presence of 0.5 microM cyclosporin A did not prevent hypercontracture but increased the percentage of cells recovering Ca2+ homeostasis to 56.2+/-3.6% and contractile function to 52+/-4.3%. CONCLUSIONS: Reperfusion-induced hypercontracture, and loss of Ca2+ homeostasis and contractile function are initiated following mitochondrial re-energization. The hypercontracture requires the production of oxidative ATP but not Ca2+ influx during reperfusion. Loss of Ca2+ homeostasis and contractile function are linked to Ca2+ influx during reperfusion, probably via opening of mitochondrial permeability transition pores.

Diazoxide causes early activation of cardiac sarcolemmal KATP channels during metabolic inhibition by an indirect mechanism.

OBJECTIVE: We have used isolated myocytes to investigate the effects of diazoxide on sarcolemmal KATP channel (sarcoKATP) activity and action potential failure during metabolic inhibition, and the role of these channels in protection of functional recovery on reperfusion. MATERIALS AND METHODS: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion. Functional recovery was assessed from the ability of cells to contract on electrical stimulation and to recover calcium homeostasis, measured with fura-2. Action potentials and KATP currents were measured using patch clamp. RESULTS: Pretreatment with diazoxide (100 microM, 5 min) increased the proportion of cells that recovered contractile function after MI and reperfusion from 16.8 +/- 2.4% to 65.0 +/- 2.2% (p<0.001) and the proportion of cells in which [Ca2+]i recovered to <250 nM. Pretreatment also accelerated action potential and contractile failure during MI. In cell-attached patches, MI activated sarcoKATP channels after 224 +/- 11 s, and diazoxide pretreatment decreased this to 145 +/- 24 s (p<0.01). However, diazoxide present in the patch pipette did not accelerate sarcoKATP channel activation. Intracellular Mg2+ rose earlier in diazoxide-pretreated cells. The sarcoKATP blocker HMR 1883 delayed action potential failure and reduced diazoxide protection. CONCLUSIONS: Diazoxide pretreatment increases recovery of function and [Ca2+]i following reperfusion. Protection is coupled with early action potential failure, due to early activation of sarcoKATP channels during metabolic inhibition (MI), which is likely to involve an indirect effect of diazoxide.

The origin of calcium overload in rat cardiac myocytes following metabolic inhibition with 2,4-dinitrophenol.

We have investigated the characteristics of the rise in cytoplasmic calcium that occurs when rat isolated cardiac ventricular myocytes are exposed to 2,4-dinitrophenol using conventional and confocal fluorescence microscopy and patch clamp. 2,4-dinitrophenol (200 microM) caused cytoplasmic calcium to increase in two phases: (1) an initial rise in fluo-3 fluorescence of 36+/-2% that was maintained until rigor contraction; (2) a further progressive rise so that fluo-3 fluorescence had increased by 177+/-12% 535 s after 2,4-dinitrophenol addition. Both phases were unaffected by removal of external Ca(2+). 2,4-dinitrophenol caused mitochondrial depolarization, measured using tetramethyl rhodamine ethyl ester fluorescence. Mitochondrial depolarization was associated with a decrease in intra-mitochondrial calcium measured with rhod-2, and experiments on myocytes loaded with both fluo-3 and rhod-2 showed that fluo-3 fluorescence increased as rhod-2 fluorescence fell. The correlation of the onset of the second phase of the increase in cytoplasmic calcium with rigor suggested that this phase was consequent on ATP depletion. DNP also caused activation of an ATP-sensitive potassium current. Depletion of sarcoplasmic reticulum calcium stores by pretreatment with ryanodine, thapsigargin and caffeine prior to the addition of 2,4-dinitrophenol did not affect the initial increase in cytoplasmic calcium, but abolished phase 2. Our results suggest that the initial rise in cytoplasmic calcium seen on application of 2,4-dinitrophenol results from release of mitochondrial calcium because of mitochondrial depolarization, while the second phase is caused by progressive release of calcium from the sarcoplasmic reticulum following depletion of intracellular ATP.