Cardioprotection through a PKC-dependent decrease in myofilament ATPase

Activation of myocardial kappa-opioid receptor-protein kinase C (PKC) pathways may improve postischemic contractile function through a myofilament reduction in ATP utilization. To test this, we first examined the effects of PKC inhibitors on kappa-opioid receptor-dependent cardioprotection. The kappa-opioid receptor agonist U50,488H (U50) increased postischemic left ventricular developed pressure and reduced postischemic end-diastolic pressure compared with controls. PKC inhibitors abolished the cardioprotective effects of U50. To determine whether kappa-opioid-PKC-dependent decreases in Ca2+-dependent actomyosin Mg2+-ATPase could account for cardioprotection, we subjected hearts to three separate actomyosin ATPase-lowering protocols. We observed that moderate decreases in myofibrillar ATPase were equally cardioprotective as kappa-opioid receptor stimulation. Immunoblot analysis and confocal microscopy revealed a kappa-opioid-induced increase in myofilament-associated PKC-epsilon, and myofibrillar Ca2+-independent PKC activity was increased after kappa-opioid stimulation. This PKC-myofilament association led to an increase in troponin I and C-protein phosphorylation. Thus we propose PKC-epsilon activation and translocation to the myofilaments causes a decrease in actomyosin ATPase, which contributes to the kappa-opioid receptor-dependent cardioprotective mechanism.     

mitoKATP通道参与心肌缺血预处理保护作用的机制

目的：探讨血管紧张素转换酶抑制剂（ACEI）和阈下缺血预处理联合预处理诱导的心肌保护作用中mi-toKatp通道激动后的作用机制：方法：采用离体大鼠心脏Langendorff灌流模型,观察心脏电脱耦联发生时间、细胞膜Na^＋／K^＋-ATPase和Ca^2＋/Mg^2＋-ATPase活性的改变：结果：单独使用卡托普利、或给予大鼠心脏2min缺血／10min复灌作为阈下缺血预处理,均不能改善长时间缺血／复灌引起的心脏收缩功能下降？而卡托普利和阂下缺血预处理联合使用可增高心脏收缩功能。mitoKatp通道特异性阻断剂5-HD可取消这一联合预处理的作用一联合预处理可引起缺血后电脱耦联发生时间延长,缺血心肌细胞膜Na^＋／K^＋-ATPase和Ca^2＋/Mg^2＋-ATPase活性增高;5-HD可取消此作用结论：mitoKatp通道参与了联合预处理延迟缺血引起的细胞间脱耦联和促进细胞膜离子通道稳定性维持的作用。     

Fasudil prevents KATP channel-induced improvement in postischemic functional recovery

Whereas activation of ATP-dependent potassium (K(ATP)) channels greatly improves postischemic myocardial recovery, the final effector mechanism for K(ATP) channel-induced cardioprotection remains elusive. RhoA is a GTPase that regulates a variety of cellular processes known to be involved with K(ATP) channel cardioprotection. Our goal was to determine whether the activity of a key rhoA effector, rho kinase (ROCK), is required for K(ATP) channel-induced cardioprotection. Four groups of perfused rat hearts were subjected to 36 min of zero-flow ischemia and 44 min of reperfusion with continuous measurements of mechanical function and (31)P NMR high-energy phosphate data: 1) untreated, 2) pinacidil (10 microM) to activate K(ATP) channels, 3) fasudil (15 microM) to inhibit ROCK, and 4) both fasudil and pinacidil. Pinacidil significantly improved postischemic mechanical recovery [39 +/- 16 vs. 108 +/- 4 mmHg left ventricular diastolic pressure (LVDP), untreated and pinacidil, respectively]. Fasudil did not affect reperfusion LVDP (41 +/- 13 mmHg) but completely blocked the marked improvement in mechanical recovery that occurred with pinacidil treatment (54 +/- 15 mmHg). Substantial attenuation of the postischemic energetic recovery was also observed. These data support the hypothesis that ROCK activity plays a role in K(ATP) channel-induced cardioprotection.     

Overexpression of A(3) adenosine receptors decreases heart rate,preserves energetics,and protects ischemic hearts

To determine whether A(3) adenosine receptor (A(3)AR) signaling modulates myocardial function, energetics, and cardioprotection, hearts from wild-type and A(3)AR-overexpressor mice were subjected to 20-min ischemia and 40-min reperfusion while (31)P NMR spectra were acquired. Basal heart rate and left ventricular developed pressure (LVDP) were lower in A(3)AR-overexpressor hearts than wild-type hearts. Ischemic ATP depletion was delayed and postischemic recoveries of contractile function, ATP, and phosphocreatine were greater in A(3)AR-hearts. To determine the role of depressed heart rate and to confirm A(3)AR-specific signaling, hearts were paced at 480 beats/min with or without 60 nmol/l MRS-1220 (A(3)AR-specific inhibitor) and then subjected to ischemia-reperfusion. LVDP was similar in paced A(3)AR-overexpressor and paced wild-type hearts. Differences in ischemic ATP depletion and postischemic contractile and energetic dysfunction remained in paced A(3)AR-overexpressor hearts versus paced wild-type hearts but were abolished by MRS-1220. In summary, A(3)AR overexpression decreased basal heart rate and contractility, preserved ischemic ATP, and decreased postischemic dysfunction. Pacing abolished the decreased contractility but not the ATP preservation or cardioprotection. Therefore, A(3)AR overexpression results in cardioprotection via a specific A(3)AR effect, possibly involving preservation of ATP during ischemia.     

Modest actomyosin energy conservation increases myocardial postischemic function

We have proposed that pharmacological preconditioning, leading to PKC-epsilon activation, in hearts improves postischemic functional recovery through a decrease in actomyosin ATPase activity and subsequent ATP conservation. The purpose of the present study was to determine whether moderate PKC-independent decreases in actomyosin ATPase are sufficient to improve myocardial postischemic function. Rats were given propylthiouracil (PTU) for 8 days to induce a 25% increase in beta-myosin heavy chain with a 28% reduction in actomyosin ATPase activity. Recovery of postischemic left ventricular developed pressure (LVDP) was significantly higher in PTU-treated rat hearts subjected to 30 min of global ischemia than in control hearts: 57.9 +/- 6.2 vs. 32.6 +/- 5.1% of preischemic values. In addition, PTU-treated hearts exhibited a delayed onset of rigor contracture during ischemia and a higher global ATP content after ischemia. In the second part of our study, we demonstrated a lower maximal actomyosin ATPase and a higher global ATP content after ischemia in human troponin T (TnT) transgenic mouse hearts. In mouse hearts with and without a point mutation at F110I of human TnT, recovery of postischemic LVDP was 55.4 +/- 5.5 and 62.5 +/- 14.5% compared with 20.0 +/- 2.9% in nontransgenic mouse hearts after 35 min of global ischemia. These results are consistent with the hypothesis that moderate decreases in actomyosin ATPase activity result in net ATP conservation that is sufficient to improve postischemic contractile function.     

Kappa 阿片受体的抗缺血性心脏保护作用--信息机制

有证据表明,心脏细胞产生强腓肽和强腓肽类多肽,它们是kappa阿片受体(κ-0R)的激动剂。κ-0R是心脏一种优势的阿片受体,其激活可改变在体和离体心脏的功能。在正常和病理情况下,内源性κ-阿片肽可能通过自分泌或旁分泌的方式调节心脏功能。心肌缺血是导致心脏功能紊乱的一个常见原因,主要表现为心肌功能减弱,心律失常及心肌梗塞等。心肌缺血时,交感神经发放增强,从而增加作功负荷及氧消耗量;而这又使缺血引发的状况更为恶化。机体抵抗缺血引发心肌损害／心律失常的保护机制之一是抑制β-肾上腺素受体(β—AR)的兴奋。κ-0R确实能抑制β-AR的激动。这种抑制主要是由于GS蛋白受到抑制,也在较小程度上由于信息通路的腺苷酸环化酶的抑制。因为该种酶能通过对百日咳毒素敏感的G蛋白转导β—AR的激动。另一保护心肌对抗缺血性损害的机制是预处理。预处理是指预先受到缺血等损伤使心脏对随后更严重的损伤产生较强的耐受能力。这种保护作用可以在预处理后即时产生,也可延至预处理后1—3天。在采用缺血或其产生的后果之一——代谢抑制作为预处理而致的心脏保护中,κ-OR参与媒介预处理的作用。用κ—OR的特异性激动剂U50488H激活κ—OR(U50488H药理性预处理,UP)可激活蛋白激酶C(PKC),开放ATY敏感的钾通道(KATP channels)及增加热休克蛋白(HSP)的产生。阻断PKC的作用,关闭KATP通道或抑制HSP的合成,均可消除UP的心脏保护作用。这些发现表明,PKC、KATP通道和HSP在UP的心脏保护中均具重要作用。此外,UP也能减低缺血造成心肌损害的因素之一,即Ca^2 的超负荷。这个事实表明UP发挥心脏保护作用至少部分地是通过减低Ca^2 的超负荷。最有趣的是,以阻断剂阻塞KATP通道,在消除UP的延迟性心脏保护作用的同时也降低了UP对Ca^2 超负荷的抑制作用。这个事实揭示了KATP通道开放所致的心脏保护作用至少部分地可能是由于防止或减低了Ca^2 的超负荷。     

Role for PKC in the adenosine-induced decrease in shortening velocity of rat ventricular myocytes

We previously demonstrated that both adenosine receptor activation and direct activation of protein kinase C (PKC) decrease unloaded shortening velocity (V(max)) of rat ventricular myocytes. The goal of this study was to further investigate a possible link among adenosine receptors, phosphoinositide-PKC signaling, and V(max) in rat ventricular myocytes. We determined that the adenosine receptor agonist R-phenylisopropyladenosine (R-PIA, 100 microM) and the alpha-adrenergic receptor agonist phenylephrine (Phe, 10 microM) increased turnover of inositol phosphates. PKC translocation from the cytosol to the sarcolemma was used as an indicator of PKC activation. Western blot analysis demonstrated an increased PKC-epsilon translocation after exposure to R-PIA, Phe, and the PKC activators dioctanoylglycerol (50 microM) and phorbol myristate acetate (1 microM). PKC-alpha, PKC-delta, and PKC-zeta did not translocate to the membrane after R-PIA exposure. Finally, PKC inhibitors blocked R-PIA-induced decreases in V(max) as well as Ca(2+)-dependent actomyosin ATPase in rat ventricular myocytes. These results support the conclusions that adenosine receptors activate phosphoinositide-PKC signaling and that adenosine receptor-induced PKC activation mediates a decrease in V(max) in ventricular myocytes.     

Contractility and ischemic response of hearts from transgenic mice with altered sarcolemmal K(ATP) channels

The functional significance of ATP-sensitive K(+) (K(ATP)) channels is controversial. In the present study, transgenic mice expressing a mutant Kir6.2, with reduced ATP sensitivity, were used to examine the role of sarcolemmal K(ATP) in normal cardiac function and after an ischemic or metabolic challenge. We found left ventricular developed pressure (LVDP) was 15-20% higher in hearts from transgenics in the absence of cardiac hypertrophy. beta-Adrenergic stimulation caused a positive inotropic response from nontransgenic hearts that was not observed in transgenic hearts. Decreasing extracellular Ca(2+) decreased LVDP in hearts from nontransgenics but not in those from transgenics. These data suggest an increase in intracellular [Ca(2+)] in transgenic hearts. Additional studies have demonstrated hearts from nontransgenics and transgenics have a similar postischemic LVDP. However, ischemic preconditioning does not improve postischemic recovery in transgenics. Transgenic hearts also demonstrate a poor recovery after metabolic inhibition. These data are consistent with the hypothesis that sarcolemmal K(ATP) channels are required for development of normal myocardial function, and perturbations of K(ATP) channels lead to hearts that respond poorly to ischemic or metabolic challenges.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The presence of two (Na+ + K+)-ATPase inhibitors in equine muscle ATP: vanadate nad a dithioerythritol-dependent inhibitor.

A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 micrometer in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate is increased by free Mg2+. The inhibition is half maximally reversed by 250 micrometer epinephrine. Equine muscle ATP was also found to contain a second (Na+ + K+)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 micrometer epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na+ + K+)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na+ + K+)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase.     

Correlation between microsomal (Na+ + K+)-ATPase activity and [3H]ouabain binding to heart tissue homogenates.

ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples.     

Comparison between alpha-adrenergic- and K-opioidergic-mediated inositol (1,4,5)P3/inositol (1,3,4,5) P4 formation in adult cultured rat ventricular cardiomyocytes

In adult cultured rat ventricular cardiac myocytes, both the alpha-adrenergic agonist phenylephrine and the selective kappa opioid receptor ligand U-50, 488H affected phosphoinositide turnover. Phenylephrine, over a time course of 10 min, caused a transient increase in Ins(1,4,5)P3 which peaked at 1 min and had returned to control at 2 min. In addition, phenylephrine produced a progressive and sustained increase in the formation of Ins (1,3,4,5)P4 which achieved a plateau after 5 min of exposure to the agonist. U-50,488H induced an increase in Ins(1,4,5)P3 which peaked at 1 min at a level significantly higher than that due to phenylephrine and was still elevated after 10 min exposure to the kappa opioid receptor agonist. In addition, U-50,488H caused a sustained increase in Ins(1,3,4,5)P4 which was comparable to that due to phenylephrine. The stimulatory effects produced by phenylephrine and U-50,488H were receptor-mediated events, since they were fully antagonized by their respective antagonists, phentolamine or Mr-1452.     

Inhibitory effects of TRK-820 on systemic skin scratching induced by morphine in rhesus monkeys

The inhibitory effects of kappa-opioid receptor agonists on systemic skin scratching induced by the intravenous administration of morphine, a micro-opioid receptor agonist, were investigated in rhesus monkeys. Intravenous pretreatment with kappa-opioid receptor agonists, either TRK-820 at 0.25 and 0.5 microg/kg or U-50488H at 64 and 128 microg/kg, inhibited systemic skin scratching induced by morphine at 1 mg/kg, i.v. in a dose-dependent manner. By the intragastric route, apparent inhibitory effects on morphine-induced systemic skin scratching were evident following pretreatment with TRK-820 at 4 microg/kg but not with U-50488H from 512 to 2048 microg/kg. These results suggest that TRK-820 produces antipruritic effects on i.v. morphine-induced systemic skin scratching and is more readily absorbed intragastrically than is U-50488H, resulting in high bioavailability in the intragastric route.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Eosin, a fluorescent marker for the high-affinity ATP site of (K+ + H+)-ATPase

Eosin has been used as a fluorescent probe for studying conformational states in (K+ + H+)-ATPase. The eosin fluorescence level is increased by Mg2+ (K0.5 = 0.2 mM). This increase is counteracted by K+ (I0.5 = 1.3 mM) and choline (I0.5 = 17.2 mM) and by ATP. Binding studies with eosin indicate that the increase and decrease in fluorescence is due to changes in binding of eosin to the enzyme. The Mg2+-induced specific binding has a Kd of 0.7 microM and a maximal capacity of 3.5 nmol per mg enzyme, which is equivalent to 2.5 site per phosphorylation site. These experiments and the fact that eosin competitively inhibits (K+ + H+)-ATPase towards ATP, suggest that eosin binds to ATP binding sites.     

High-affinity calcium-stimulated, magnesium-dependent adenosine triphosphatase in Trypanosoma cruzi.

1. A high-affinity (Ca2+ + Mg2+)-ATPase and a low-affinity Mg(2+)-ATPase were identified in the 105,000 g fraction from epimastigote forms of Trypanosoma cruzi, the agent of Chagas' disease (Tulahuen strain). 2. Activities were conserved after enzyme solubilization with deoxycholate. 3. The Ca(2+)-stimulated ATPase activity was (a) lower than that of the Mg(2+)-ATPase; (b) inhibited by p-chloromercurobenzoate and orthovanadate and (c) insensitive to oligomycin. 4. Optimal stimulation by Ca2+ was observed at pH 6.5-6.8 in the presence of 1 mM MgCl2 and 0.1 M KCl. 5. The Mg(2+)-ATPase was insensitive to p-chloromercurobenzoate and orthovanadate and did not require KCl for activity. 6. Kinetic analysis of the (Ca2+ + Mg2+)-ATPase yielded a half-maximal stimulating concentration of 1.1 microM for Ca2+ and a Km of 66 microM for ATP. 7. The (Ca2+ + Mg2+)-ATPase clearly differed from the Ca(2+)- or Mg(2+)-ATPases previously characterized in the same strain of T. cruzi (Frasch et al., 1978; Comp. Biochem. Physiol. 60B, 271-275).     

Characterisation of a high affinity Ca2+-stimulated, Mg2+-dependent ATPase in the rat parotid plasma membrane

Two Ca2+-stimulated ATPase activities have been identified in the plasma membrane of rat parotid: (a) a (Ca2+ + Mg2+)-ATPase with high affinity for free Ca2+ (apparent Km = 208 nM, Vmax = 188 nmol/min per mg) and requiring micromolar concentration of Mg2+ and (b) a (Ca2+ or Mg2+)-ATPase with relatively low affinity for free Ca2+ (K0.5 = 23 microM) or free Mg2+ (K0.5 = 26 microM). The low-affinity (Ca2+ or Mg2+)-ATPase can be maximally stimulated by Ca2+ alone or Mg2+ alone. The high-affinity (Ca2+ + Mg2+)-ATPase exhibits sigmoidal kinetics with respect to ATP concentration with K0.5 = 0.4 mM and a Hill coefficient of 1.91. It displays low substrate specificity with respect to nucleotide triphosphates. Although trifluoperazine inhibits the activity of the high affinity (Ca2+ + Mg2+)-ATPase only slightly, it inhibits the activity of the low-affinity (Ca2+ or Mg2+)-ATPase quite potently with 22 microM trifluoperazine inhibiting the enzymic activity by 50%. Vanadate, inositol 1,4,5-trisphosphate, phosphatidylinositol 4,5-bisphosphate, Na+,K+ and ouabain had no effect on the activities of both ATPases. Calmodulin added to the plasma membranes does not stimulate the activities of both ATPases. The properties of the high-affinity (Ca2+ + Mg2+)-ATPase are distinctly different from those of the previously reported Ca2+-pump activity of the rat parotid plasma membrane.     

Finding of a KCl-independent, electrogenic, and ATP-driven H+-pumping activity in rat light gastric membranes and its effect on the membrane K+ transport activity

Resting rat light gastric membranes prepared through 2H2O and Percoll gradient centrifugations were enriched not only with (H+-K+)-ATPase and K+ transport activity (Im, W. B., Blakeman, D. P., and Davis, J. P. (1985) J. Biol. Chem. 260, 9452-9460), but also with a K+-independent, ATP-dependent H+-pumping activity. This intravesicular acidification has been ascribed to an oligomycin-insensitive H+-ATPase which differed from (H+-K+)-ATPase in several respects. The H+-ATPase is electrogenic, apparently of lower capacity, required a lower optimal ATP concentration (4 microM for the H+-ATPase and 500 microM for (H+-K+)-ATPase), of lower sensitivity to vanadate and sulfhydryl agents such as p-chloromercuribenzoate and N-ethylmaleimide, and insensitive to SCH 28,080, a known competitive inhibitor of (H+-K+)-ATPase with respect to K+. Operation of the H+-ATPase, however, appeared to interfere with the K+ transport activity in the light gastric membranes, probably through development of intravesicular positive membrane potential; for example, micromolar levels of Mg2+-ATP fully inhibited K+ uptake and stimulated K+ efflux as measured with 86Rb+. Involvement of (H+-K+)-ATPase in the K+ transport is not likely, since the inhibitory effect of Mg2+-ATP continued even after removal of the nucleotide with an ATP-scavenging system. Moreover, nigericin, an electroneutral H+/K+ exchanger, could bypass the inhibitory effect of Mg2+-ATP and equilibrate the membrane vesicles with 86Rb+ while valinomycin, an electrogenic K+ ionophore, could not. Finally, the H+-ATPase could possibly be involved in the acid secretory process, since its H+-pumping activity was removed from the light gastric membrane fraction upon carbachol treatment, along with the K+ transport and (H+-K+)-ATPase activities. We have speculated that the H+-ATPase is responsible for maintaining the K+-permeable intracellular membrane vesicles acidic and K+ free during the resting state of acid secretion and may contribute to basal acid secretion.     

Protection of cardiac myocytes via delta(1)-opioid receptors, protein kinase C, and mitochondrial K(ATP) channels

The objective of the present study was to investigate the role of delta(1)-opioid receptors in mediating cardioprotection in isolated chick cardiac myocytes and to investigate whether protein kinase C and mitochondrial ATP-sensitive K(+) (K(ATP)) channels act downstream of the delta(1)-opioid receptor in mediating this beneficial effect. A 5-min preexposure to the selective delta(1)-opioid receptor agonist (-)-TAN-67 (1 microM) resulted in less myocyte injury during the subsequent prolonged ischemia compared with untreated myocytes. 7-Benzylidenenaltrexone, a selective delta(1)-opioid receptor antagonist, completely blocked the cardioprotective effect of (-)-TAN-67. Naltriben methanesulfonate, a selective delta(2)-opioid receptor antagonist, had only a slight inhibitory effect on (-)-TAN-67-mediated cardioprotection. Nor-binaltorphimine dihydrochloride, a kappa-opioid receptor antagonist, did not affect (-)-TAN-67-mediated cardioprotection. The protein kinase C inhibitor chelerythrine and the K(ATP) channel inhibitors glibenclamide, a nonselective K(ATP) antagonist, and 5-hydroxydecanoic acid, a mitochondrial selective K(ATP) antagonist, reversed the cardioprotective effect of (-)-TAN-67. These results suggest that the delta(1)-opioid receptor is present on cardiac myocytes and mediates a potent cardioprotective effect via protein kinase C and the mitochondrial K(ATP) channel.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.