Arachidonic acid metabolism in the human placenta: identification of a putative lipoxygenase.

Arachidonic acid (ARA) metabolites maintain pregnancy and control parturition. We generated a network of 77 proteins involved in placental ARA metabolism to identify novel proteins in this pathway. We identified a long pathway within this network which showed that secretory and cytosolic phospholipase A2 proteins act in concert. The functions of all network proteins expressed in the placental decidua were determined by database searches. Thus ARA metabolism was linked to carbohydrate metabolism. One protein, transmembrane protein 62 (TMEM62), expressed in decidua was previously uncharacterized, and was identified as a putative lipoxygenase. TMEM62 may play a role in pregnancy and/or parturition.

Increased protein synthesis is necessary for the development of late preconditioning against myocardial stunning.

In phase I of this study, the rate of protein synthesis was measured by the incorporation of [(3)H]leucine into the protein pool in the heart of conscious rabbits. At 2 h after ischemic preconditioning (PC) with six 4-min occlusion/4-min reperfusion (O/R) cycles (group II), the [3H]leucine content in the ischemic-reperfused region was increased by 82% compared with that in controls (group I), indicating increased protein synthesis. This increase was completely abrogated by pretreatment with cycloheximide (CH; group III). In phase II, rabbits underwent six O/R cycles for three consecutive days (days 1-3). Controls (group IV) exhibited late PC against myocardial stunning on days 2 and 3. In group V, which received CH 30 min before the 1st O/R cycle on day 1 (same dose as group III), late PC against stunning on day 2 was completely abrogated. In group VI, pretreatment with CH 24 h before the 1st sequence of O/R cycles had no effect on myocardial stunning on day 1, indicating that the absence of late PC on day 2 in group V cannot be ascribed to delayed toxicity of CH. Taken together, these results demonstrate that, in the conscious rabbit, ischemic PC causes a rapid increase in myocardial protein synthesis and that this increased protein synthesis (or at least a fraction of it) is necessary for the development of the protection against myocardial stunning 24 h later. The late phase of ischemic PC is therefore dependent on the formation of new proteins in intact animals.

Nitric oxide synthase is the mediator of late preconditioning against myocardial infarction in conscious rabbits.

BACKGROUND: Despite intense investigation, the effector of the infarct-limiting protection observed during the late phase of ischemic preconditioning (PC) remains unknown. The goal of this study was to test the hypothesis that late PC against myocardial infarction is mediated by the activity of nitric oxide synthase (NOS). METHODS AND RESULTS: Conscious rabbits underwent a 30-minute coronary occlusion followed by 3 days of reperfusion. In group I (control group, n= 10), infarct size (tetrazolium staining) averaged 56.8+/-5.3% of the risk region, which was decreased to 27.6+/-2.5% (P<0.05) in rabbits preconditioned 24 hours earlier with a sequence of six 4-minute occlusion/4-minute reperfusion cycles (group II, n= 10). When preconditioned rabbits were given the nonselective NOS inhibitor N(omega)-nitro-L-arginine (L-NA, 13 mg/kg i.v. [group III, n=8]) or the selective iNOS inhibitor aminoguanidine (AG, 150 mg/kg SC [group V, n=7]) before the 30-minute occlusion, the protective effect of late PC was completely abrogated; that is, infarct size (59.9+/-4.5% and 65.8+/-3.3%, respectively) was similar to that measured in the control group. Measurements of systolic wall thickening (sonomicrometry) demonstrated that L-NA and AG also abolished the improved recovery of myocardial function effected by late PC in group II. When rabbits were given L-NA or AG without prior PC (group IV [n=8] and group VI [n=6], respectively), infarct size did not differ from that observed in controls (53.8+/-4.3% and 59.8+/-4.3%, respectively), demonstrating that L-NA and AG do not increase the extent of cell death in nonpreconditioned myocardium. CONCLUSIONS: Taken together, these results indicate that in the conscious rabbit, the infarct-sparing effect of the late phase of ischemic PC is mediated by the activity of NOS and suggest that the specific isoform primarily responsible for this cardioprotective phenomenon is iNOS. Thus, NO appears to be a pivotal component of the pathophysiological cascade of late PC.

The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase. Evidence that nitric oxide acts both as a trigger and as a mediator of the late phase of ischemic preconditioning.

Seventy-four conscious rabbits undergoing a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3) were assigned to nine groups. In group I (controls, n = 8), the recovery of systolic wall thickening (WTh) after the sixth reperfusion was markedly improved on days 2 and 3 compared with day 1, indicating late preconditioning (PC) against myocardial stunning; the total deficit of WTh after the sixth reperfusion was reduced by 56% on day 2 and 50% on day 3 compared with day 1 (P < .01). Administration on day 2 of the nonselective NO synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NA) (group II, n = 8) or of the selective inducible NOS inhibitors aminoguanidine (AG) (group IV, n = 8) and S-methylisothiourea sulfate (SMT) (group VI, n = 6) completely abrogated late PC against stunning on day 2. On day 3, the expected PC effect became manifest in all groups. Administration of L-NA, AG, or SMT on day 1 (groups III [n = 7], V [n = 6], and VII [n = 5], respectively) had no discernible effect on the deficit of WTh on day 1, indicating that these agents do not augment the severity of myocardial stunning in nonpreconditioned myocardium. In group VIII (n = 7), the abrogation of late PC by SMT on day 2 was completely reversed by the concomitant administration of L-arginine (595 mg/kg IV), indicating that it was not due to nonspecific NOS-unrelated actions. Administration of L-arginine alone on day 2 (group IX [n = 5]) had no effect on the deficit of WTh. Furthermore, administration of L-NA on day 1 (group III) prevented the appearance of the PC effect on day 2, whereas AG (group V) and SMT (group VI) did not, suggesting that the development of late PC on day 1 is triggered by the endothelial (type III) isoform of NOS. This study demonstrates that three structurally different NOS inhibitors (L-NA, AG, and SMT), given 24 hours after the PC ischemia, consistently abrogate late PC against myocardial stunning in conscious rabbits, indicating that this cardioprotective effect is mediated by the activity of NOS. The results obtained with AG and SMT specifically implicate the inducible (type II) isoform as the mediator of the protection on day 2. Previous studies have shown that NO triggers the development of late PC. The present results indicate that NO plays a dual role in late PC against stunning, acting initially as the trigger and subsequently as the mediator of the protection.

Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxide.

Recent studies in conscious pigs and rabbits have demonstrated that a series of brief coronary occlusions renders the heart relatively resistant to myocardial "stunning" 24 hours later (late preconditioning [PC] against stunning). The mechanism of this powerful cardioprotective response is unknown. The goal of the present study was to test the hypothesis that the development of late PC against stunning is triggered by increased generation of NO during the first ischemic challenge. Conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). On day 1, rabbits received either an intravenous infusion of the NO synthase inhibitor NG-nitro-L-arginine (L-NA, 13 mg/kg before the first occlusion) (group II, n = 10) or vehicle (group I [control], n = 10). In the control group, on day 1 systolic wall thickening (WTh) in the ischemic/reperfused region remained significantly depressed for 4 hours after the sixth reperfusion, indicating myocardial stunning. On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 60% on day 2 and 55% on day 3 compared with day 1 (P < .01). In the L-NA-treated group, the total deficit of WTh on day 1 was similar to that observed in the control group. On day 2, however, the total deficit of WTh was not significantly different from that observed on day 1 and was 132% greater than that observed in control rabbits on day 2 (P < .01). On day 3, the total deficit of WTh was 66% less than that noted on day 2 (P < .01). Thus, in L-NA-treated rabbits the sequence of six coronary occlusions and reperfusions performed on day 1 failed to precondition against stunning on day 2, but the same sequence performed on day 2 did precondition against stunning on day 3. Another group of rabbits (group III, n = 6) received L-NA on day 1 in the absence of ischemia and was subjected to the occlusion/ reperfusion sequence on days 2 and 3. In these animals, the total deficit of WTh on day 2 did not differ from that observed in control rabbits on day 1, indicating that administration of L-NA did not exacerbate the severity of myocardial stunning 24 hours later; therefore, the absence of late PC against stunning on day 2 in group II cannot be ascribed to a delayed deleterious action of L-NA on WTh. In conclusion, these results demonstrate that the NO synthase inhibitor L-NA completely blocks the development of late PC against myocardial stunning in conscious rabbits, indicating that NO generated as a result of the PC ischemia triggers the development of the cardioprotective response observed 24 hours later. NO is known to exert numerous biological actions resulting in rapid but transient physiological responses. The present observations support a novel pathophysiological paradigm in which NO also plays a key role in the delayed myocardial adaptations to ischemic stress, acting as a signaling step in the transduction pathway that leads to increased resistance to subsequent ischemic injury.

Nitric oxide triggers late preconditioning against myocardial infarction in conscious rabbits.

We tested the hypothesis that late preconditioning (PC) against myocardial infarction is triggered by the formation of nitric oxide (NO). Conscious rabbits underwent a 30-min coronary occlusion followed by 3 days of reperfusion. In group I (control group, n = 10), rabbits were not preconditioned, whereas in group II (n = 10), they were preconditioned 24 h earlier with a sequence of six 4-min occlusion/4-min reperfusion cycles. Myocardial infarct size (tetrazolium staining) was reduced by 50% by PC (28.6 +/- 3.2% of the risk region in group II vs. 56.9 +/- 5.9% in controls, P < 0.05). This reduction in cell death was associated with improved recovery of myocardial function [systolic thickening fraction (by sonomicrometry) at 3 days: 2.0 +/- 11.0% of baseline in group II vs. -20.0 +/- 2.8% in group I, P < 0.05]. Group III rabbits (n = 11) underwent the same protocol as group II except that the rabbits received the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA, 13 mg/kg) before the PC ischemia. In these animals, infarct size did not differ significantly from that observed in control rabbits, indicating that L-NNA completely blocked the development of late PC against myocardial infarction. In group IV (n = 9), rabbits received L-NNA as in group III, but without the six occlusion-reperfusion cycles, and were subjected to the 30-min occlusion 24 h later. In this group, infarct size did not differ from that observed in controls, demonstrating that pretreatment with L-NNA, in itself, did not affect the extent of cell death. Taken together, these results indicate that, in the conscious rabbit, the development of late PC against myocardial infarction is triggered by the generation of NO during the PC ischemia. It is proposed that NO plays a key role in the delayed myocardial adaptation to ischemic stress.