Kappa-opioid receptor stimulation increases the expression of Na+-H+ exchange gene in the heart

Kappa-opioid receptor (OR) stimulation increases intracellular pH (pHi) via activating the Na+-H+ exchange (NHE). In the present study, we determined the expression of the gene of NHE1, the predominant NHE isoform in the heart, and intracellular pH (pHi) upon kappa-OR stimulation in the rat heart. We found that 1 microM U50,488H (trans-3,4-dichloro-N-methyl-N-(2-(1 pyrrolidinyl)cyclohexyl)benzeneacetamide), a selective kappa-OR agonist, increased the expression of the NHE1 gene. We also found that U50,488H dose-dependently increased pHi in the heart. The effects were abolished by 1 microM nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, indicating that the events were kappa-OR mediated. The effects on both NHE1 gene expression and pHi were also abolished by 5 microM chelerythrine and 5 microM BSM (bisyndolylmaleimide), protein kinase C (PKC) inhibitors, indicating that PKC mediated the actions. In addition, the effect of U50,488H on pHi was blocked by 10 microM EIPA (ethylisopropyl amiloride), a NHE1 inhibitor, indicating that NHE1 also mediated the action of U50,488H. The present study provides evidence for the first time that kappa-OR stimulation increased the NHE1 gene expression in the heart via a PKC dependent pathway. Kappa-OR stimulation also increases pHi via PKC and NHE in the heart.     

Role of endogenous opioids in ischemic preconditioning but not in short-term hibernation in pigs

Endogenous opioids are involved in ischemic preconditioning (IP) in several species. Whether or not opioids are important for IP and short-term myocardial hibernation (STMH) in pigs is currently unknown. In 34 enflurane-anesthetized pigs, the left anterior descending coronary artery was flow constantly perfused. Subendocardial blood flow (Endo), infarct size (IS; percent area at risk), and the free energy change of ATP hydrolysis (DeltaG) were determined. After 90-min severe ischemia and 120-min reperfusion, IS averaged 28.3 +/- 5.4% (means +/- SE) (n = 8; Endo: 0.047 +/- 0.009 ml. min(-1) x g(-1)). IP by 10-min ischemia and 15-min reperfusion reduced IS to 9.9 +/- 3.8% (P < 0.05, n = 8; Endo: 0.044 +/- 0.009 ml. min(-1) x g(-1)). After naloxone (1 mg/kg iv followed by 2 microg x kg(-1) x min(-1)), IS averaged 25.8 +/- 7.0% (n = 6; Endo: 0.039 +/- 0.008 ml x min(-1) x g(-1)) without and 24.7 +/- 4.7% (n = 6; Endo: 0.044 +/- 0.006 ml x min(-1) x g(-1)) with IP. At 5-min moderate ischemia in the presence of naloxone, Endo decreased from 0.90 +/- 0.07 to 0.28 +/- 0.03 ml x min(-1) x g(-1)and DeltaG decreased from -58.6 +/- 1.0 to -52.6 +/- 0.4 kJ/mol. Prolongation of ischemia to 90 min did not alter Endo, but DeltaG recovered toward control values (57.7 +/- 1.1 kJ/mol), and the myocardium remained viable. These responses are identical to those of nonnaloxone-treated pigs. Endogenous opioids are involved in IP but not in STMH in pigs.     

Platelet-activating factor induces cardioprotection in isolated rat heart akin to ischemic preconditioning: role of phosphoinositide 3-kinase and protein kinase C activation

Ischemic preconditioning (IP) is a cardioprotective mechanism against myocellular death and cardiac dysfunction resulting from reperfusion of the ischemic heart. At present, the precise list of mediators involved in IP and the pathways of their mechanisms of action are not completely known. The aim of the present study was to investigate the role of platelet-activating factor (PAF), a phospholipid mediator that is known to be released by the ischemic-reperfused heart, as a possible endogenous agent involved in IP. Experiments were performed on Langendorff-perfused rat hearts undergoing 30 min of ischemia followed by 2 h of reperfusion. Treatment with a low concentration of PAF (2 x 10(-11) M) before ischemia reduced the extension of infarct size and improved the recovery of left ventricular developed pressure during reperfusion. The cardioprotective effect of PAF was comparable to that observed in hearts in which IP was induced by three brief (3 min) periods of ischemia separated by 5-min reperfusion intervals. The PAF receptor antagonist WEB-2170 (1 x 10(-9) M) abrogated the cardioprotective effect induced by both PAF and IP. The protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M) or the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (5 x 10(-5) M) also reduced the cardioprotective effect of PAF. Western blot analysis revealed that following IP treatment or PAF infusion, the phosphorylation of PKC-epsilon and Akt (the downstream target of PI3K) was higher than that in control hearts. The present data indicate that exogenous applications of low quantities of PAF induce a cardioprotective effect through PI3K and PKC activation, similar to that afforded by IP. Moreover, the study suggests that endogenous release of PAF, induced by brief periods of ischemia and reperfusion, may participate to the triggering of the IP of the heart.     

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。     

Protein kinase C is involved in cardioprotective effects of ischemic preconditioning on infarct size and ventricular arrhythmia in rats in vivo

Protein kinase C (PKC) has been known to play an important role in ischemic preconditioning (IP). This study was designed to examine whether the translocation of PKC is associated with the cardioprotective effects of IP in vivo on infarct size and ventricular arrhythmias in a rat model.Using anesthetized rats, heart rate, systolic blood pressure, infarct size and ventricular arrhythmias during 45 min of coronary occlusion were measured. PKC activity was assayed in both the cytosolic and cell membrane fraction . Brief 3-min periods of ischemia followed by 10 min of reperfusion were used to precondition the myocardium. Calphostin C was used to inhibit PKC.Infarct size was significantly reduced by IP (68.1 (2.5)%, mean (S.E.) vs. 45.2 (3.4)%, p < 0.01). The reduction in infarct size by IP was abolished by pretreatment with calphostin C. The total number of ventricular premature complex (VPC) during 45 min of coronary occlusion was reduced by IP (1474 (169) beats/45 min vs. 256 (82) beats/45 min, p < 0.05). The reduction the total number of VPC induced by IP was abolished by the administration of calphostin C before the episode of brief ischemia. The same tendency was observed in the duration of ventricular tachycardia and the incidence of ventricular fibrillation. PKC activity in the cell membrane fraction transiently increased immediately after IP (100 vs. 142%, p < 0.01) and returned to baseline 15 min after IP. Pretreatment with calphostin C prevented the translocation of PKC.The translocation of PKC plays an important role in the cardioprotective effect of IP on infarct size and ventricular arrhythmias in anesthetized rats.     

Preconditioning blocks cardiocyte apoptosis: role of K(ATP) channels and PKC-epsilon

The aims of this study were to determine whether preconditioning blocks cardiocyte apoptosis and to determine the role of mitochondrial ATP-sensitive K(+) (K(ATP)) channels and the protein kinase C epsilon-isoform (PKC-epsilon) in this effect. Ventricular myocytes from 10-day-old chick embryos were used. In the control series, 10 h of simulated ischemia followed by 12 h of reoxygenation resulted in 42 +/- 3% apoptosis (n = 8). These results were consistent with DNA laddering and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Preconditioning, elicited with three cycles of 1 min of ischemia separated by 5 min of reoxygenation before subjection to prolonged simulated ischemia, markedly attenuated the apoptotic process (28 +/- 4%, n = 8). The selective mitochondrial K(ATP) channel opener diazoxide (400 micromol/l), given before ischemia, mimicked preconditioning effects to prevent apoptosis (22 +/- 4%, n = 6). Pretreatment with 5-hydroxydecanoate (100 micromol/l), a selective mitochondrial K(ATP) channel blocker, abolished preconditioning (42 +/- 2%, n = 6). In addition, the effects of preconditioning and diazoxide were blocked with the specific PKC inhibitors G?-6976 (0.1 micromol/l) or chelerythrine (4 micromol/l), given at simulated ischemia and reoxygenation. Furthermore, preconditioning and diazoxide selectively activated PKC-epsilon in the particulate fraction before simulated ischemia without effect on the total fraction, cytosolic fraction, and PKC delta-isoform. The specific PKC activator phorbol 12-myristate 13-acetate (0.2 micromol/l), added during simulated ischemia and reoxygenation, mimicked preconditioning to block apoptosis. Opening mitochondrial K(ATP) channels blocks cardiocyte apoptosis via activating PKC-epsilon in cultured ventricular myocytes. Through this signal transduction, preconditioning blocks apoptosis and preserves cardiac function in ischemia-reperfusion.     

Role of delta-opioid receptor agonists on infarct size reduction in swine

Opioids are involved in cardiac ischemic preconditioning. Important species differences in cellular signaling mechanisms, antiarrhythmic, and antistunning effects have been described. The role of the delta-opioid receptor activation in swine remains unknown. Forty minutes before a 45-min occlusion and 180-min reperfusion of the left anterior descending coronary artery, open-chest, pentobarbital-anesthetized swine received either 1) saline (controls); 2) [D-Ala(2),D-Leu(5)]enkephalin (DADLE); 3) [D-Pen(2,5)]enkephalin (DPDPE); 4) deltorphin-D, a novel delta(2)-opioid agonist; or 5) ischemic preconditioning (IP). Assessed were 1) infarct size to area at risk (IS, triphenyltetrazolium staining), 2) regional and global myocardial function (sonomicrometry, ventricular pressure catheters), and 3) arrhythmias (electrocardiogram analyses). It was found that DPDPE and deltorphin-D pretreatment reduced IS from 64.7 +/- 5 to 36.5 +/- 6% and 27.4 +/- 11% (P < 0.01), respectively, whereas DADLE had no effect (66.8 +/- 3%). Both IP and DADLE had a proarrhythmic effect (P < 0.01). However, no differences in global or regional myocardial function or arrhythmia scores were observed between groups. This suggests that delta-receptor-specific opioids provide cardioprotection in swine.     

Essential activation of PKC-delta in opioid-initiated cardioprotection

Stimulation of the delta(1)-opioid receptor confers cardioprotection to the ischemic myocardium. We examined the role of protein kinase C (PKC) after delta-opioid receptor stimulation with TAN-67 or D-Ala(2)-D-Leu(5)-enkephalin (DADLE) in a rat model of myocardial infarction induced by a 30-min coronary artery occlusion and 2-h reperfusion. Infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of the area at risk (IS/AAR). Control animals, subjected to ischemia and reperfusion, had an IS/AAR of 59.9 +/- 1.8. DADLE and TAN-67 administered before ischemia significantly reduced IS/AAR (36.9 +/- 3.9 and 36.7 +/- 4.7, respectively). The delta(1)-selective opioid antagonist 7-benzylidenenaltrexone (BNTX) abolished TAN-67-induced cardioprotection (54.4 +/- 1.3). Treatment with the PKC antagonist chelerythrine completely abolished DADLE- (61.8 +/- 3.2) and TAN-67-induced cardioprotection (55.4 +/- 4.0). Similarly, the PKC antagonist GF 109203X completely abolished TAN-67-induced cardioprotection (54.6 +/- 6.6). Immunofluorescent staining with antibodies directed against specific PKC isoforms was performed in myocardial biopsies obtained after 15 min of treatment with saline, chelerythrine, BNTX, or TAN-67 and chelerythrine or BNTX in the presence of TAN-67. TAN-67 induced the translocation of PKC-alpha to the sarcolemma, PKC-beta(1) to the nucleus, PKC-delta to the mitochondria, and PKC-epsilon to the intercalated disk and mitochondria. PKC translocation was abolished by chelerythrine and BNTX in TAN-67-treated rats. To more closely examine the role of these isoforms in cardioprotection, we utilized the PKC-delta selective antagonist rottlerin. Rottlerin abolished opioid-induced cardioprotection (48.9 +/- 4.8) and PKC-delta translocation without affecting the translocation of PKC-alpha, -beta(1), or -epsilon. These results suggest that PKC-delta is a key second messenger in the cardioprotective effects of delta(1)-opioid receptor stimulation in rats.     

内源性阿片物质参与大鼠缺血预处理的心肌保护作用

实验以离体灌流的ＳＤ大鼠心脏为模型,用ｋ特异性拮抗剂的ＭＲ２２６６研究ｋ阿片受体的阻断与缺血预处理的关系,用放射免疫分析法研究ＩＰ及长时间缺血对心肌强啡肽Ａ１－１３浓度的影响,探索Ｋ阿片物质在ＩＰ过程中的作用和地位。     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Role of kappa-opioid receptor activation in pharmacological preconditioning of swine

Pharmacological preconditioning with kappa-opioid receptor agonists is proarrhythmic and exerts antipreconditioning effects in rats. In swine, it is unknown whether kappa-opioid receptor stimulation plays a role in pharmacological preconditioning. Swine were preconditioned with 1) saline (controls), 2) [d-Ala(2),d-Leu(5)]enkephalin (DADLE), 3) morphine, 4) pentazocine, 5) norbinaltorphimine (nor-BNI), 6) DADLE + nor-BNI, 7) morphine + nor-BNI, or 8) pentazocine + nor-BNI before occlusion (45 min) and reperfusion (180 min) of the left anterior descending coronary artery. Infarct size to area at risk (IS), regional (systolic shortening) and global (pressures and flows) myocardial function, and arrhythmia occurrence were assessed. Only DADLE + nor-BNI preconditioning significantly decreased infarct size compared with controls (47 +/- 13 vs. 65 +/- 5%, P < 0.05); morphine preconditioning was not cardioprotective with or without kappa-opioid receptor blockade (nor-BNI). DADLE preconditioning significantly increased ischemia-induced arrhythmias relative to controls, whereas pentazocine-preconditioned animals (n = 2) experienced intractable ventricular fibrillation during ischemia. kappa-Opioid receptor blockade with DADLE or pentazocine preconditioning alleviated proarrhythmic effects. These results suggest that kappa-opioid receptor activation during pharmacological preconditioning is proarrhythmic in swine.     

Role of mitochondrial and sarcolemmal K(ATP) channels in ischemic preconditioning of the canine heart

We tested whether mitochondrial or sarcolemmal ATP-sensitive K(+) (K(ATP)) channels play a key role in ischemic preconditioning (IP) in canine hearts. In open-chest beagle dogs, the left anterior descending artery was occluded four times for 5 min each with 5-min intervals of reperfusion (IP), occluded for 90 min, and reperfused for 6 h. IP as well as cromakalim and nicorandil (nonspecific K(ATP) channel openers) markedly limited infarct size (6.3 +/- 1.2, 8.9 +/- 1.9, and 7.2 +/- 1.6%, respectively) compared with the control group (40.9 +/- 4.1%). A selective mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate, partially blunted the limitation of infarct size in the animals subjected to IP and those treated with cromakalim and nicorandil (21.6 +/- 3.8, 25.1 +/- 4.6, and 19.8 +/- 5.2%, respectively). A nonspecific K(ATP) channel blocker, glibenclamide, completely abolished the effect of IP (38.5 +/- 6.2%). Intracoronary or intravenous administration of a mitochondria-selective K(ATP) channel opener, diazoxide, at >100 micromol/l could only partially decrease infarct size (19.5 +/- 4.3 and 20.1 +/- 4.4%, respectively). In conclusion, mitochondrial and sarcolemmal K(ATP) channels independently play an important role in the limitation of infarct size by IP in the canine heart.     

Ischemic preconditioning protects cardiomyocytes against ischemic injury by inducing GRP78

Ischemic preconditioning (IP) conferred by brief ischemia-reperfusion induces resistance to cell injury due to the following lethal ischemia. This study aimed to elucidate whether 78-kDa glucose-regulated protein (GRP78), a main ER molecular chaperone, contributes to IP-mediated protection against ischemic myocardial injury. In a rat coronary artery occlusion model, the GRP78 protein level increased to 210% of the sham level by early IP with three cycles of 4-min ischemia and 4-min reperfusion. The IP reduced infarct size in subsequent lethal ischemia. In primary cardiomyocytes, the simulated IP procedure, incubation in oxygen-glucose deprivation (OGD) medium, also increased the GRP78 expression and suppressed the cell death caused by lethal ischemia. Transfection of grp78 antisense oligonucleotide attenuated the IP-mediated resistance to ischemia. This study showed for the first time that early IP up-regulates myocardial GRP78. It was suggested that GRP78 induced by early IP contributes to protect cardiomyocytes against ischemic injury.     

Cardioprotective effects of acute and chronic opioid treatment are mediated via different signaling pathways

A 5-day exposure to morphine exerts a profound cardioprotective phenotype in murine hearts. In the present study, we examined mechanisms by which morphine generates this effect, exploring the roles of G(i) and G(s) proteins, PKA, PKC, and beta-adrenergic receptors (beta-AR) in acute and chronic opioid preconditioning. Langendorff-perfused hearts from placebo, acute morphine (AM; 10 micromol/l)-, or chronic morphine (CM)-treated mice (75-mg pellet, 5 days) underwent 25-min ischemia and 45-min reperfusion. After reperfusion, placebo-treated hearts exhibited marked contractile and diastolic dysfunction [rate-pressure product (RPP), 40 +/- 4% baseline; end-diastolic pressure (EDP), 33 +/- 3 mmHg], whereas AM hearts showed significant improvement in recovery of RPP and EDP (60 +/- 3% and 23 +/- 4 mmHg, respectively; P < 0.05 vs. placebo). Furthermore, CM hearts demonstrated a complete return of diastolic function and significantly greater recovery of contractile function (83 +/- 3%, P < 0.05 vs. both placebo and AM). Pretreatment with G(i) protein inhibitor pertussis toxin abolished AM protection while partially attenuating CM recovery (P < 0.05 vs. placebo). Treatment with G(s) inhibitor NF-449 did not affect AM preconditioning yet completely abrogated CM preconditioning. Similarly, PKA inhibition significantly attenuated the ischemia-tolerant state afforded by CM, whereas it was ineffective in AM hearts. PKC inhibition with chelerythrine was ineffective in CM hearts while completely abrogating AM preconditioning. Moreover, whereas beta(1)-AR blockade with CGP-20712A failed to alter recovery in CM hearts, the beta(2)-AR antagonist ICI-118,551 significantly attenuated postischemic recovery. These data describe novel findings whereby CM preconditioning is mediated by a PKC-independent pathway involving PKA, beta(2)-AR, and G(s) proteins, whereas AM preconditioning is mediated via G(i) proteins and PKC.     

Bradykinin induces a rapid release of inositol trisphosphate from a neuroblastoma hybrid cell line NCB-20 that is not antagonized by enkephalin

In mouse neuroblastoma x Chinese hamster brain clonal cell line NCB-20, bradykinin (BK) receptor stimulation causes phosphoinositide hydrolysis and release of inositol phosphates. Maximum stimulation (4-fold) of [2-3H]inositol trisphosphate (IP3) release in the absence of Li+ from NCB-20's prelabelled for 20-24 hours with [2-3H]myo-inositol (15 microCi/confluent 60mm dish) occurred after 5-10 seconds of bradykinin exposure, with an EC50 of approximately 100nM. Inositol bisphosphate (IP2) and inositol monophosphate (IP1) also showed increases (2.9-fold and 1.5 fold, respectively), with peaks at 15-20 seconds and 50 seconds, respectively. Under these same conditions, D-Ala2-D-Leu5 enkephalin (DADLE) (10 microM), an opiate agonist with 2nM affinity, gave no stimulation of IP3 release. Furthermore, it did not block BK-initiated release, both when applied simultaneously with BK and when cells were preincubated with DADLE for 100 minutes to lower cyclic AMP levels. These results show that pain-inducing BK has a major acute stimulatory effect on receptor-phospholipase C-coupled IP3 release, the opioid peptide DADLE has no such effect and, DADLE does not block the IP3 release induced by BK.     

Atrial natriuretic peptide relaxes arterial basal tone induced by coarctation hypertension.

We have investigated the vasorelaxant effect of atrial natriuretic peptide (ANP) on isolated non-contracted aorta from coarctation hypertensive rats (HR) and the role of endothelium in this vasorelaxant action. After 7-14 days of surgery, mean blood pressure was higher (P < 0.01) in HR compared with sham operated rats (SR), used as the control. ANP (10(-6) mol/l) significantly lowered basal tone in previously unstimulated HR thoracic aortic rings; however, it had no effect in HR abdominal aorta or in SR abdominal and thoracic aorta. Endothelial destruction potentiated the vasorelaxant effect of ANP on basal tone in HR thoracic aorta. A similar potentiation of the ANP-response was observed by pre-treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) mol/l) or methylene blue (2 x 10(-5) mol/l) in unrubbed HR thoracic aorta. Treatment with calcium-free Krebs + EGTA (2 x 10(-3) mol/l) + sodium nitroprusside (10(-5) mol/l) or calcium-free Krebs significantly decreased basal tone and abolished ANP-response. These effects were observed only in HR thoracic aorta. Similarly, staurosporine (10(-7) mol/l) and calphostin C (10(-6) mol/l), inhibitors of protein kinase C (PKC), diminished basal tone and abolished the ANP-response in HR thoracic aorta. Acetylcholine (10(-6) mol/l) had a small but significant action on the basal tone of unrubbed HR thoracic aorta. These results demonstrate that ANP has a vasorelaxant effect on aortic basal tone when the vessel is exposed to high blood pressure. Inhibition of ANP effects on basal tone by calcium-free Krebs and PKC antagonists suggests that the HR aorta increases Ca2+-active tone, that modifies the response of vascular smooth muscle to the vasodilating hormone ANP.     

Role of protein phosphatases in hypoxic preconditioning

To find a protein kinase C (PKC)-independent preconditioning mechanism, hypoxic preconditioning (HP; i.e., 10-min anoxia and 10-min reoxygenation) was applied to isolated rat hearts before 60-min global ischemia. HP led to improved recovery of developed pressure and reduced end-diastolic pressure in the left ventricle during reperfusion. Protection was unaffected by the PKC inhibitor bisindolylmaleimide (BIM; 1 micromol/l). It was abolished by the inhibitor of protein phosphatases 1 and 2A cantharidin (20 or 5 micromol/l) and partially enhanced by the inhibitor of protein phosphatase 2A okadaic acid (5 nmol/l). In adult rat cardiomyocytes treated with BIM and exposed to 60-min simulated ischemia (anoxia, extracellular pH 6.4), HP led to attenuation of anoxic Na(+)/Ca(2+) overload and of hypercontracture, which developed on reoxygenation. This protection was prevented by treatment with cantharidin but not with okadaic acid. In conclusion, HP exerts PKC-independent protection on ischemic-reperfused rat hearts and cardiomyocytes. Protein phosphatase 1 seems a mediator of this protective mechanism.     

Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning

We examined whether the mitochondrial ATP-sensitive K channel (K(ATP)) is an effector downstream of protein kinase C-epsilon (PKC-epsilon) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 +/- 6.8% of the left ventricle to 20.3 +/- 3.7%. PC significantly increased PKC-epsilon protein in the particulate fraction from 51 +/- 4% of the total to 60 +/- 4%, whereas no translocation was observed for PKC-delta and PKC-alpha. In mitochondria separated from the other particulate fractions, PC increased the PKC-epsilon level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) blocker, after PC abolished the cardioprotection of PC, whereas PKC-epsilon translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial K(ATP) opener, infused 10 min before ischemia limited infarct size to 5.2 +/- 1.4%, but this agent neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. Together with the results of earlier studies showing mitochondrial K(ATP) opening by PKC, the present results suggest that mitochondrial K(ATP)-mediated cardioprotection occurs subsequent to PKC-epsilon activation by PC.     

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.