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.     

Ischemic preconditioning in rats: role of mitochondrial K(ATP) channel in preservation of mitochondrial function

We examined the role of the sarcolemmal and mitochondrial K(ATP) channels in a rat model of ischemic preconditioning (IPC). Infarct size was expressed as a percentage of the area at risk (IS/AAR). IPC significantly reduced infarct size (7 +/- 1%) versus control (56 +/- 1%). The sarcolemmal K(ATP) channel-selective antagonist HMR-1098 administered before IPC did not significantly attenuate cardioprotection. However, pretreatment with the mitochondrial K(ATP) channel-selective antagonist 5-hydroxydecanoic acid (5-HD) 5 min before IPC partially abolished cardioprotection (40 +/- 1%). Diazoxide (10 mg/kg iv) also reduced IS/AAR (36.2 +/- 4.8%), but this effect was abolished by 5-HD. As an index of mitochondrial bioenergetic function, the rate of ATP synthesis in the AAR was examined. Untreated animals synthesized ATP at 2.12 +/- 0.30 micromol x min(-1) x mg mitochondrial protein(-1). Rats subjected to ischemia-reperfusion synthesized ATP at 0.67 +/- 0.06 micromol x min(-1) x mg mitochondrial protein(-1). IPC significantly increased ATP synthesis to 1.86 +/- 0.23 micromol x min(-1) x mg mitochondrial protein(-1). However, when 5-HD was administered before IPC, the preservation of ATP synthesis was attenuated (1.18 +/- 0.15 micromol x min(-1) x mg mitochondrial protein(-1)). These data are consistent with the notion that inhibition of mitochondrial K(ATP) channels attenuates IPC by reducing IPC-induced protection of mitochondrial function.     

Adenosine and opioid receptor-mediated cardioprotection in the rat: evidence for cross-talk between receptors

The relative roles of free-radical production, mitochondrial ATP-sensitive K+ (mitoKATP) channels and possible receptor cross-talk in both opioid and adenosine A1 receptor (A1AR) mediated protection were assessed in a rat model of myocardial infarction. Sprague-Dawley rats were subjected to 30 min of occlusion and 90 min of reperfusion. The untreated rats exhibited an infarct of 58.8 +/- 2.9% [infarct size (IS)/area at risk (AAR), %] at the end of reperfusion. Pretreatment with either the nonselective opioid receptor agonist morphine or the selective A1AR agonist 2-chloro-cyclopentyladenosine (CCPA) dramatically reduced IS/AAR to 41.1 +/- 2.2% and 37.9 +/- 5.5%, respectively (P < 0.05). Protection afforded by either morphine or CCPA was abolished by the reactive oxygen species scavenger N-(2-mercaptopropionyl)glycine or the mitoKATP channel blocker 5-hydroxydecanoate. Both morphine- and CCPA-mediated protection were attenuated by the selective A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine and the selective delta1-opioid receptor (DOR) antagonist 7-benzylidenealtrexone. Simultaneous administration of morphine and CCPA failed to enhance the infarct-sparing effect of either agonist alone. These data suggest that both DOR and A1AR-mediated cardioprotection are mitoKATP and reactive oxygen species dependent. Furthermore, these data suggest that there are converging pathways and/or receptor cross-talk between A1AR- and DOR-mediated cardioprotection.     

Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation

Opioids introduced at reperfusion (R) following ischemia (I) reduce infarct size much like postconditioning, suggesting the hypothesis that postconditioning increases cardiac opioids and activates local opioid receptors. Anesthetized male rats subjected to 30 min regional I and 3 h R were postconditioned with three cycles of 10 s R and 10 s reocclusion at onset of R. Naloxone (NL), its peripherally restricted analog naloxone methiodide, delta-opioid receptor (DOR) antagonist naltrindole (NTI), kappa-opioid receptor antagonist norbinaltorphimine (NorBNI), and mu-opioid receptor (MOR) antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were administered intravenously 5 min before R. The area at risk (AAR) was comparable among groups, and postconditioning reduced infarct size from 57 +/- 2 to 42 +/- 2% (P < 0.05). None of the antagonists alone altered infarct size. All antagonists abrogated postconditioning protection at higher doses. However, blockade of infarct sparing by postconditioning was lost, since tested doses of NL, NTI, NorBNI, and CTAP were lowered. The efficacy of NorBNI declined first at 3.4 micromol/kg, followed sequentially by NTI (1.1), NL (0.37), and CTAP (0.09), suggesting likely MOR and perhaps DOR participation. Representative small, intermediate, and large enkephalins in the AAR were quantified (fmol/mg protein; mean +/- SE). I/R reduced proenkephalin (58 +/- 9 vs. 33 +/- 4; P < 0.05) and sum total of measured enkephalins, including proenkephalin, peptide B, methionine-enkephalin, and methionine-enkephalin-arginine-phenylalanine (139 +/- 17 vs. 104 +/- 7; P < 0.05) compared with shams. Postconditioning increased total enkephalins (89 +/- 8 vs. 135 +/- 5; P < 0.05) largely by increasing proenkephalin (33 +/- 4 vs. 96 +/- 7; P < 0.05). Thus the infarct-sparing effect of postconditioning appeared to involve endogenously activated MORs and possibly DORs, and preservation of enkephalin precursor synthesis in the AAR.     

COX-2 and iNOS in opioid-induced delayed cardioprotection in the intact rat

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) have been previously implicated in the late phase of cardioprotection associated with opioid-induced and ischemic preconditioning (IPC) in conscious rabbits and COX-2 in isolated rat hearts pretreated with an exogenous delta opioid agonist. However, it is not know if both iNOS and COX-2 mediate the late phase of cardioprotection induced by opioids in the intact blood-perfused rat. Therefore, we investigated the role of COX-2 and iNOS in the delayed phase of protection mediated by delta opioid receptor activation. Rats were pretreated 24 hours prior to an occlusion/reperfusion protocol with the selective non-peptide delta opioid agonists, BW373U86 (BW) and SNC-121 (SNC). NS-398, a selective COX-2 inhibitor was administered after the 24-hour recovery period just prior to index ischemia. The selective iNOS inhibitors, S-methylthiourea (SMT) and aminoguanidine (AG), were administered in conjunction with opioid pretreatment or were also given 24 hours after opioid administration just prior to index ischemia. COX-2 inhibition by NS-398 given 24 hours after opioid administration attenuated the protective effects of both BW and SNC (46 +/- 6 vs. 13 +/- 3 and 51 +/- 5 vs. 29 +/- 2, p < 0.001, respectively). Similarly, inhibition of iNOS following 24 hours of treatment with opioids also attenuated the protective effects of BW and SNC. However, the delayed protective effects of the opioids were not attenuated by pretreatment with the iNOS inhibitors 24 hours prior to the infarct protocol. These results suggest that both COX-2 and iNOS are mediators of delayed protection induced by non-peptide delta opioid agonists. It appears that the trigger effect is not dependent on the activity of iNOS or COX-2 but the late phase of cardioprotection is dependent on the upregulation of these enzymes.     

Stress-activated protein kinase phosphorylation during cardioprotection in the ischemic myocardium

Stress-activated protein kinases may be essential to cardioprotection. We assessed the role of p38 in an in vivo rat model of ischemia-reperfusion. Ischemic preconditioning (IPC) and the delta(1)-opioid receptor agonist 2-methyl-4aalpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha-octahydroquinolino [2,3,3-g]isoquinoline (TAN-67) significantly reduced infarct size (IS), expressed as a percentage of the area at risk (AAR), versus animals subjected only to 30 min of ischemia and 2 h of reperfusion (7.1 +/- 1.5 and 29.6 +/- 3.3 vs. 59.7 +/- 1.6%). The p38 antagonist SB-203580 attenuated IPC when it was administered before (34.0 +/- 6.9%) or after (25.0 +/- 3.8%) the IPC stimulus; however, it did not significantly attenuate TAN-67-induced cardioprotection (39.6 +/- 3.2). We also assessed the phosphorylation of p38 and c-jun NH(2)-terminal kinase (JNK) throughout ischemia-reperfusion in nuclear and cytosolic fractions. After either intervention, no increase was detected in the phosphorylation state of either enzyme in the nuclear fraction or for p38 in the cytosolic fraction versus control hearts. However, there was a robust increase in JNK activity in the cytosolic fraction immediately on reperfusion that was more pronounced in animals subjected to IPC or administered TAN-67. These data suggest that SB-203580 likely attenuates IPC via the inhibition of kinases other than p38, which may include JNK. The data also suggest that activation of JNK during early reperfusion may be an important component of cardioprotection.     

Beneficial actions of nalorphine during hemorrhagic shock in cats

Endogenous opiates have been reported to have detrimental effects on the circulatory system during hemorrhagic shock. However, the specific opiate receptor subtype which mediates these actions has not been defined. In the present study, we have utilized the mixed agonist/antagonist, nalorphine (N-allylnormorphine), which exhibits kappa (kappa) and sigma (sigma) receptor agonism as well as mu (mu) receptor antagonism, to investigate the role of the mu receptor in hemorrhagic shock. Nalorphine (2 mg/kg) produced no significant changes in any observed experimental variable in sham-shocked animals. Shocked animals treated with nalorphine (2 mg/kg) maintained significantly higher final mean arterial blood pressures (MABP) than animals which received only vehicle (102 +/- 3.8 vs 61 +/- 6.6 mm Hg, respectively, p less than 0.001). In addition, nalorphine significantly reduced the rise in plasma MDF activity observed in untreated hemorrhaged animals (42 +/- 3.0 vs 59 +/- 4 U/ml, p less than 0.02). Our results support a significant role for the mu receptor in the deleterious actions of endogenous opioids during hemorrhagic shock.     

Heat shock-induced cardioprotection activates cytoskeletal-based cell survival pathways

To define better the subcellular mechanism of heat shock (HS)-induced cardioprotection, we examined the effect of HS, as well as selective expression of individual HS proteins (HSPs), on cell injury in neonatal rat ventricular myocytes (NRVM). HS was induced in NRVM by a rapid elevation of temperature to 42 degrees C for 20 min followed by 20-24 h of recovery at 37 degrees C. Other NRVM were infected with a replication-deficient adenovirus encoding HSP27 or HSP70. On the same day, all groups were subjected to metabolic inhibition (MI). Cell injury was assayed by measurement of the percentage of total lactate dehydrogenase released, the percentage of cells staining with trypan blue, or TdT-mediated dUTP nick-end labeling, whereas cell signaling was assayed by immunoblot analysis and coimmunoprecipitation. Before MI, the viability of all treated groups did not differ significantly from control NRVM. HS resulted in a significant increase in HSP70 and HSP27 expression. Infection with either virus caused a significant increase in selective HSP content compared with control NRVM. HS protected NRVM from injury. Selective expression of HSP27 or HSP70 alone was not protective in NRVM, but dual infection with both viral vectors (HSP27 + HSP70) was protective. HS and HSP27 + HSP70 expression caused increased paxillin localization in the membrane fraction, which persisted in response to MI, compared with control NRVM. HS increased the integrin-paxillin-focal adhesion kinase interaction, whereas targeted inhibition of focal adhesion kinase activity abolished the integrin-paxillin association and resulted in an increase in cell death. HS and HSP27 + HSP70 expression increased the association of members of the focal adhesion complex and protected NRVM against irreversible injury. Cytoskeletal-based signaling pathways at focal adhesion junctions may represent a unique pathway of cardioprotection.     

Aging, opioid-receptor agonists and antagonists, and the vestibulosympathetic reflex in humans.

Animal studies indicate that opioids inhibit the firing rate of vestibular neurons, which are important in mediating the vestibulosympathetic reflex. Furthermore, this inhibition appears to be greater in more mature rats. In the present study, we tested the hypotheses that opioids inhibit the vestibulosympathetic reflex in humans and that endogenous opioids contribute to the age-related impairment of the vestibulosympathetic reflex. These hypotheses were tested by measuring muscle sympathetic nerve activity (MSNA), arterial blood pressure, and heart rate responses to otolith organ engagement during head-down rotation (HDR) in young (24 +/- 2 yr old) and older (63 +/- 2 yr) subjects before and after administration of either an opioid-receptor antagonist (16 mg naloxone in 9 young and 8 older subjects) or an opioid-receptor agonist (60 mg codeine in 7 young and 7 older subjects). Naloxone did not augment the reflex increase in MSNA during HDR in young (Delta7 +/- 2 vs. Delta4 +/- 2 bursts/min and Delta81 +/- 23 vs. Delta60 +/- 24% change in burst frequency and total MSNA before and after naloxone, respectively) or older subjects (Delta2 +/- 2 vs. Delta1 +/- 2 burst/min and Delta8 +/- 7 vs. Delta8 +/- 9% before and after naloxone). Similarly, codeine did not attenuate the increase in MSNA during HDR in young (Delta8 +/- 1 vs. Delta7 +/- 2 bursts/min and Delta53 +/- 4 vs. Delta64 +/- 16% before and after codeine) or older subjects (Delta6 +/- 4 vs. Delta3 +/- 3 bursts/min and Delta38 +/- 21 vs. Delta33 +/- 20%). Mean arterial blood pressure and heart rate responses to HDR were not altered by either naloxone or codeine. These data do not provide experimental support for the concept that opioids modulate the vestibulosympathetic reflex in humans. Moreover, endogenous opioids do not appear to contribute the age-associated impairment of the vestibulosympathetic reflex.     

Heat shock gene expression in Xenopus laevis A6 cells in response to heat shock and sodium arsenite treatments

Continuous exposure of a Xenopus laevis kidney epithelial cell line, A6, to either heat shock (33 degrees C) or sodium arsenite (50 microM) resulted in transient but markedly different temporal patterns of heat-shock protein (HSP) synthesis and HSP 70 and 30 mRNA accumulation. Heat-shock-induced synthesis of HSPs was detectable within 1 h and reached maximum levels by 2-3 h. While sodium arsenite induced the synthesis of some HSPs within 1 h, maximal HSP synthesis did not occur until 12 h. The pattern of HSP 70 and 30 mRNA accumulation was similar to the response observed at the protein level. During recovery from heat shock, a coordinate decline in HSPs and HSP 70 and 30 mRNA was observed. During recovery from sodium arsenite, a similar phenomenon occurred during the initial stages. However, after 6 h of recovery, HSP 70 mRNA levels persisted in contrast to the declining HSP 30 mRNA levels. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of 5 HSPs in the HSP 70 family, of which two were constitutive, and 16 different stress-inducible proteins in the HSP 30 family. In conclusion, heat shock and sodium arsenite induce a similar set of HSPs but maximum synthesis of the HSP is temporally separated by 12-24 h.     

高频电刺激股神经减小大鼠心肌缺血-再灌注后的梗塞范围

通过氨基甲酸乙酯麻醉大鼠观察股神经电刺激对缺血- 再灌注心肌的影响,旨在证实外周神经刺激对心肌有无保护效应,并明确其可能的作用机制。心肌缺血区和梗塞区分别用伊文思蓝和氯化硝基四氮唑蓝染色确定,心肌梗塞范围定义为心肌梗塞区重量占心肌缺血区重量的百分比。所得结果如下:(1)在心肌缺血30 min 和再灌注120 min 过程中,梗塞心肌占缺血心肌的(54.96±0.82)%。 高频电刺激(10 V,100 Hz,1ms)股神经5 min 可使心肌梗塞范围减少到(36.94±1.34)% (P<0.01), 表明 (2)高频电刺激股神经对缺血-再灌注心肌有保护作用。然而,低频电刺激(10 V, 10 Hz, 1 ms)股神经对缺血-再灌注心肌梗塞范围无影响。 预先应用非选择性阿片肽受体阻断剂纳洛酮(5 mg/kg, i.v.)或非选择性KATP 通道阻断剂格列苯脲(5 mg/kg, i.v.)均能完全 (3)取消高频电刺激股神经对缺血-再灌注心肌的保护作用。以上结果提示,高频外周神经刺激可以减小缺血- 再灌注心肌的梗塞范围,其可能的作用机制是: 高频电刺激股神经时中枢神经系统内释放的内源性阿片肽和由此激活的心肌KATP通道的开放介导了这种保护作用。     

Cardioprotection and myocardial salvage by a disodium disuccinate astaxanthin derivative (Cardax)

Cardioprotection in humans by carotenoids has been inferred from observational and epidemiologic studies, however, direct studies of cardioprotection and myocardial salvage by carotenoids are lacking. In the current study, intravenous (I.V.) pre-treatment with a novel carotenoid derivative (disodium disuccinate astaxanthin; Cardax) was evaluated as a myocardial salvage agent in a Sprague-Dawley rat infarct model. Animals were dosed once per day I.V. by tail vein injection for 4 days at one of 3 doses (25, 50, and 75 mg/kg) prior to the infarct study carried out on day 5. The results were compared with control animals treated with saline vehicle. Thirty (30) minutes of occlusion of the left anterior descending (LAD) coronary artery was followed by 2 hours of reperfusion prior to sacrifice, a regimen which resulted in a mean infarct size (IS) as a percent (%) of the area at risk (AAR) of 59 +/- 3%. Area at risk was quantified by Patent blue dye injection, and infarct size (IS) was determined by triphenyltetrazolium chloride (TTC) staining. Cardax at 50 and 75 mg/kg for 4 days resulted in a significant mean reduction in IS/AAR to 35 +/- 3% (41% salvage) and 26 +/- 2% (56% salvage), respectively. Infarct size and myocardial salvage were significantly, and linearly, correlated with plasma levels of non-esterified, free astaxanthin at the end of reperfusion. These results suggest that parenteral Cardax may find utility in those clinical applications where pre-treatment of patients at risk for myocardial infarction is performed.     

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.     

Cardioprotection is strain dependent in rat in response to whole body hyperthermia

Previous results showed a genetic component to cardioprotection. Therefore, we investigated the heat shock response in Wistar and Sprague-Dawley (SD) rats at 24 and 48 h. Rats were subjected to whole body hyperthermia achieving colonic temperatures of 40 or 42 degrees C for 20 min. After recovery hearts were excised for protein measurements or were subjected to 30 min of ischemia and then 2 h of reperfusion. Heat shock protein (HSP) expression was determined by Western blotting and infarct size was determined by triphenyltetrazolium staining. All groups of SD and Wistar rats demonstrated HSP72 and HSP90 induction at both time points in response to a heat stress of 42 degrees C. At 24 h there was only a significant reduction in infarct size seen in control vs. small SD (60.0 +/- 4.8 vs. 26.5 +/- 2.3) rats. However, at 48 h control versus small SD (60.0 +/- 4.8 vs. 17.6 +/- 3.8) and Wistar (59.4 +/- 4.3 vs. 29.8 +/- 6.0) and control versus large SD (53.7 +/- 2.6 vs. 19.8 +/- 4.7) and Wistar (57.3 +/- 1.6 vs. 34.5 +/- 2.8) rats demonstrated a significant reduction in infarct size with a greater reduction observed in SD rats. We conclude that heat shock-induced cardioprotection in rats is dependent on strain, temperature, time after stress, and size.     

Effects of the selective EET antagonist, 14,15-EEZE, on cardioprotection produced by exogenous or endogenous EETs in the canine heart

Previously, we demonstrated (17) that 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) produce marked reductions in myocardial infarct size. Although it is assumed that this cardioprotective effect of the EETs is due to a specific interaction with a membrane-bound receptor, no evidence has indicated that novel EET antagonists selectively block the EET actions in dogs. Our goals were to investigate the effects of 11,12- and 14,15-EET, the soluble epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), and the putative selective EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), on infarct size of barbital anesthetized dogs subjected to 60 min of coronary artery occlusion and 3 h of reperfusion. Furthermore, the effect of 14,15-EEZE on the cardioprotective actions of the selective mitochondrial ATP-sensitive potassium channel opener diazoxide was investigated. Both 11,12- and 14,15-EET markedly reduced infarct size [expressed as a percentage of the area at risk (IS/AAR)] from 21.8 +/- 1.6% (vehicle) to 8.7 +/- 2.2 and 9.4 +/- 1.3%, respectively. Similarly, AUDA significantly reduced IS/AAR from 21.8 +/- 1.6 to 14.4 +/- 1.2% (low dose) and 9.4 +/- 1.8% (high dose), respectively. Interestingly, the combination of the low dose of AUDA with 14,15-EET reduced IS/AAR to 5.8 +/- 1.6% (P < 0.05), further than either drug alone. Diazoxide also reduced IS/AAR significantly (10.2 +/- 1.9%). In contrast, 14,15-EEZE had no effect on IS/AAR by itself (21.0 +/- 3.6%), but completely abolished the effect of 11,12-EET (17.8 +/- 1.4%) and 14,15-EET (19.2 +/- 2.4%) and AUDA (19.3 +/- 1.6%), but not that of diazoxide (10.4 +/- 1.4%). These results suggest that activation of the EET pathway, acting on a putative receptor, by exogenous EETs or indirectly by blocking EET metabolism, produced marked cardioprotection, and the combination of these two approaches resulted in a synergistic effect. These data also suggest that 14,15-EEZE is not blocking the mitochondrial ATP-sensitive potassium channel as a mechanism for antagonizing the cardioprotective effects of the EETs.     

The influence of heart disease on characteristics, quality of life, use of health resources, and costs of COPD in primary care settings

Background

Ectonucleotidase dependent adenosine generation has been implicated in preconditioning related cardioprotection against ischemia-reperfusion injury, and treatment with a soluble ectonucleotidase has been shown to reduce myocardial infarct size (IS) when applied prior to induction of ischemia. However, ectonucleotidase treatment according to a clinically applicable protocol, with administration only after induction of ischemia, has not previously been evaluated. We therefore investigated if treatment with the ectonucleotidase apyrase, according to a clinically applicable protocol, would reduce IS and microvascular obstruction (MO) in a large animal model.

Methods

A percutaneous coronary intervention balloon was inflated in the left anterior descending artery for 40 min, in 16 anesthetized pigs (40-50 kg). The pigs were randomized to 40 min of 1 ml/min intracoronary infusion of apyrase (10 U/ml, n = 8) or saline (0.9 mg/ml, n = 8), twenty minutes after balloon inflation. Area at risk (AAR) was evaluated by ex vivo SPECT. IS and MO were evaluated by ex vivo MRI.

Results

No differences were observed between the apyrase group and saline group with respect to IS/AAR (75.7 ± 4.2% vs 69.4 ± 5.0%, p = NS) or MO (10.7 ± 4.8% vs 11.4 ± 4.8%, p = NS), but apyrase prolonged the post-ischemic reactive hyperemia.

Conclusion

Apyrase treatment according to a clinically applicable protocol, with administration of apyrase after induction of ischemia, does not reduce myocardial infarct size or microvascular obstruction.     

Modulation of motilin-induced somatostatin release in dogs by naloxone

Somatostatin release in dogs is modulated by exogenous and endogenous opioids. Since postprandial somatostatin secretion is in part due to the stimulatory effect of postprandially activated gastrointestinal hormones as well as endogenous opioids, it was of interest to determine the interaction between motilin, a known stimulus of somatostatin release, and endogenous opioids with regard to activation of D-cell function. In a group of eight conscious dogs the infusion of synthetic porcine motilin at doses of 0.05, 0.25 and 0.5 micrograms/kg X hr elicited a significant increase of peripheral vein plasma somatostatin-like immunoreactivity (SLI), confirming previously reported data. The additional infusion of the opiate receptor antagonist naloxone attenuated this SLI response, suggesting that endogenous opioids participate in motilin-induced SLI release. Since previous studies have shown that the interaction between endogenous opioids and postprandial somatostatin secretion is modified by elevated plasma glucose levels, the experiments were repeated during an IV glucose (0.2 g/min) background infusion increasing circulating glucose levels by 20-30 mg/dl. During IV glucose, the SLI response to motilin was almost abolished. In this group the addition of naloxone restored the SLI response, indicating that the inhibitory effect of elevated glucose on D-cell function is, at least in part, mediated by endogenous opioids. These data suggest that motilin has to be considered as one regulatory factor which participates in the previously observed interaction between glucose and endogenous opioids during postprandial SLI release.     

Effects of naloxone on breathing movements during hypercapnia in the fetal lamb

To determine whether endogenous opioids influence the fetal breathing response to CO2 we have investigated the effect of the opiate antagonist, naloxone on the incidence, rate, and amplitude of breathing movements during hypercapnia in fetal lambs in utero. In 20 experiments in six pregnant sheep (130-145 days gestation) hypercapnia was induced by giving the ewe 4-6% CO2-18% O2 in N2 to breathe for 60 min. After 30 min of hypercapnia either naloxone (13 experiments) or saline (7 experiments) was infused intravenously for the remaining 30 min. During hypercapnia breath amplitude increased from 5.8 +/- 0.5 to 9.1 +/- 1.2 mmHg (P less than 0.001), and infusion of naloxone was associated with a further significant increase to 15.7 +/- 1.2 mmHg (P less than 0.001). Naloxone had no effect on the incidence or rate of breathing movements during hypercapnia. After hypercapnia there was a significant decrease in the incidence of fetal breathing movements in the naloxone group (14.7 +/- 3.2%). Infusion of saline during hypercapnia had no effect on incidence, rate, or amplitude of fetal breathing movements. These results suggest that endogenous opioids act to suppress or limit breath amplitude during hypercapnia but do not affect rate or incidence of breathing movements.