Cardioprotection induced by hydrogen sulfide preconditioning involves activation of ERK and PI3K/Akt pathways

We previously reported that hydrogen sulfide (H₂S) preconditioning (SP) produces cardioprotective effects against ischemia in rat cardiac myocytes. The present study aims to elucidate the signaling mechanisms involved in SP-induced cardioprotection by investigating the role of extracellular signal regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt. We found that preconditioning with NaHS (a H₂S donor) for three cycles significantly decreased myocardial infarct size and improved heart contractile function in the isolated rat hearts. NaHS (1–100 μM) concentration-dependently increased cell viability and percentage of rod-shaped cardiac myocytes. Blockade of ERK1/2 with PD 98059 or PI3K/Akt with LY-294002 or Akt inhibitor III during either preconditioning or ischemia periods significantly attenuated the cardioprotection of SP, suggesting that both ERK1/2 and PI3K/Akt triggered and mediated the cardioprotection of SP. Moreover, SP induced ERK1/2 and Akt phosphorylation in isolated hearts. The phosphorylation of ERK1/2 induced by SP was attenuated by either glibenclamide, an ATP-sensitive K⁺ channel (K_ATP) blocker, or chelerythrine, a specific protein kinase C (PKC) blocker. In addition, ischemic-preconditioning-induced ERK1/2 activation was reversed by inhibiting endogenous H₂S production, suggesting that ERK1/2 activation induced by ischemic preconditioning was, at least partly, mediated by endogenous H₂S. In conclusion, K_ATP/PKC/ERK1/2 and PI3K/Akt pathways contributed to SP-induced cardioprotection.     

Fibroblast growth factor-2-induced cardioprotection against myocardial infarction occurs via the interplay between nitric oxide,protein kinase signaling,and ATP-sensitive potassium channels

Fibroblast growth factor-2 (FGF2) protects the heart from ischemia–reperfusion (I-R) injury via a vast network of protein kinases. In the heart, downstream effectors of these FGF2-triggered signals have not yet been identified. It is hypothesized that nitric oxide (NO) signaling and ATP-sensitive potassium (K_ATP) channel activity are key effectors of protein kinases activated by FGF2-mediated cardioprotection. Hearts with a cardiac-specific overexpression of FGF2 (FGF2 Tg) were subjected to I-R injury in the absence or the presence of selective inhibitors of NO synthase (NOS) isoforms or sarcolemmal (sarcK_ATP) and mitochondrial (mitoK_ATP) K_ATP channels. Multiple NOS isoforms are necessary for FGF2-mediated cardioprotection, and nitrite levels are significantly reduced in FGF2 Tg hearts upon inhibition of protein kinase C or mitogen-activated protein kinases. Likewise, sarcK_ATP and mitoK_ATP channels are important for cardioprotection elicited by endogenous FGF2. These findings suggest that FGF2-induced cardioprotection occurs via protein kinase-NOS pathways as well as K_ATP channel activity.     

Hydrogen sulfide improves cardiomyocytes electrical remodeling post ischemia/reperfusion injury in rats

Hydrogen sulfide (H₂S), produced by cystanthionine-γ-lysase (CSE) in the cardiovascular system, is an endogenous gaseous mediator exerting pronounced physiological effects as the third gasotransmitter in addition to nitric oxide (NO) and carbon monoxide (CO). Accumulating evidence indicated that H₂S could mediate the cardioprotective effects in myocardial ischemia model. Ventricular arrhythmia is the most important risk factor for cardiac mortality and sudden death after acute myocardial infarction (AMI). The potential impact of H₂S on cardiomyocytes electrical remodeling post ischemic insult is not fully explored now. Present study investigated the role of H₂S on cardiomyocytes electrical remodeling in rats with ischemia/reperfusion injury. H₂S concentration was reduced and arrhythmia score was increased in this model. CSE mRNA level was also upregulated in the ischemic myocardium. Exposure to exogenous NaHS reduced the action potential duration (APD), inhibited L-type Ca²⁺ channels and activated K_ATP channels in cardiomyocytes isolated from ischemic myocardium Exogenous H₂S application improves electrical remodeling in cardiomyocytes isolated from ischemic myocardium. These results indicated that reduced H₂S level might be linked to ischemia/reperfusion induced arrhythmias.     

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Purpose

Prostaglandin (PG) E₂ is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE₂ on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE₂-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods

We explored the involvement of HO-1 on PGE₂ regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results

LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE₂ or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE₂ on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE₂ and enhanced by blockage of the endogenous PGE₂ effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE₂. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions

AKAP plays an important role in PGE₂ regulation of COX-2 expression, and the suppression of HO-1 by PGE₂-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.     

IL-1β Stimulates the Expression of Prostaglandin Receptor EP4 in Human Chondrocytes by Increasing Production of Prostaglandin E2

Prostaglandin (PG) E₂, which exerts its actions via the PG receptors EP1–4, is produced from arachidonic acid by cyclooxygenase (COX)-1 and COX-2. The aim of this study was to investigate the mechanisms by which interleukin (IL)-1β induces the expression of PG receptors in cultured human chondrocytes and to explore the role of PGE₂ in this process. The cells were cultured with 0, 10, or 100 U/mL IL-1β with or without 1 μM celecoxib, a specific inhibitor of COX-2, for up to 28 days. Expression of the genes encoding COX-1, COX-2, and EP1–4 was quantified using real-time PCR, and expression of the corresponding proteins was examined using immunohistochemical staining. PGE₂ production was determined using ELISA. IL-1β treatment caused a marked dose- and time-dependent increase in the levels of PGE₂, COX-2, and EP4 as compared with the untreated control. It did not affect the expression of COX-1, and it decreased the expression of EP1 and EP2. EP3 expression was not detected in either the absence or the presence of IL-1β. When celecoxib was also present, IL-1β failed to stimulate PGE₂ production and EP4 expression, but its stimulatory effect on COX-2 expression and its inhibitory effect on EP1 and EP2 expression were unchanged. IL-1β increases the production of PGE₂, COX-2, and the PG receptor EP4 in cultured human chondrocytes. The increase in EP4 expression appears to be a result of the increased PGE₂ production.     

CGRP-induced activation of KATP channels in follicular Xenopus oocytes

The two-microelectrode voltage-clamp technique was used to monitor K⁺ channel activity in Xenopus oocyte follicular cells, which are electrically coupled to the oocyte itself by gap junctions. Endogenous vasodilators such as calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), prostaglandin E₂ (PGE₂) and adenosine activate glibenclamide-ATP-sensitive K⁺ (K_ATP) channels in Xenopus oocyte follicular cells. The mechanism of action of CGRP was studied in detail. CGRP effects undergo a rapid desensitization. CGRP acts via CGRP_I receptors. Its effects are antagonized by the amino-truncated CGRP analog hCGRP(8–37). The second messenger for CGRP activation of K_ATP channels is cAMP. Phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine enhances the CGRP response while adenyl cyclase inhibition by either 2,5-dideoxyadenosine or progesterone nearly completely depresses the CGRP response. Vasoconstrictors such as ACh and angiotensin II also have receptors in follicular cells. ACh strongly inhibits the CGRP activation of K⁺ channels as it inhibits the activation of K_ATP channels by P1060, but angiotensin II does not. It is concluded that as in vascular smooth muscle cells, CGRP and probably other hyperpolarizing vasodilators open K_ATP channels in follicular cells by protein kinase A activation.Thanks are due to C. Roulinat and F. Aguila for expert technical assistance. This work was supported by the Centre National de la Recherche Scientifique (CNRS).     

磷酯酶A2抑制剂DTNB对大鼠心肌 缺血再灌注心律失常的影响

目的:研究非钙依赖性的磷脂酶A₂ (PLA₂)和ATP敏感性钾通道(K_ATP)在大鼠心肌缺血再灌注(I/R)心律失常中的作用。方法:结扎大鼠左冠状动脉前降支造成缺血10 min,然后放开再灌注10 min。在心肌缺血前5 min分别给予PLA₂抑制剂5,5'-dithio-bis(2-nitrobenzoic acid)(DTNB)(16 mg/kg),K_ATP开放剂pinacidil(0.2 mg/kg),K_ATP阻断剂glibenclamide(0.3 mg/kg)。结果:与对照组相比,DTNB可降低心律失常评分、磷酸肌酸激酶(CPK)和乳酸脱氢酶 (LDH)值(P＜0.05);而glibenclamide对I/R心律失常无影响,但升高CPK,LDH值(P＜0.05);与glibenclamide组比,在glibenclamide阻断K_ATP后,DTNB可引起致死性心律失常,并可升高LDH值(P＜0.05);pinacidil可完全抑制I/R引起的室颤和室速。结论:PLA₂抑制剂DTNB可减轻I/R损伤,表明PLA₂在I/R心律失常中起着重要作用,同时也表明激活的K_ATP具有抗I/R心律失常的作用。但DTNB对抗I/R心律失常的作用与K_ATP的关系仍有待进一步研究。     

Compressive Force Induces Osteoclast Differentiation via Prostaglandin E2 Production in MC3T3-E1 Cells

In orthodontic tooth movement, prostaglandin E₂ (PGE₂) released from osteoblasts can alter the normal process of bone remodeling. We previously showed that compressive force (CF) controls bone formation by stimulating the production of PGE₂ and Ep2 and/or Ep4 receptors in osteoblasts. The present study was undertaken to examine the effect of CF on the production of PGE₂, cyclooxygenase-2 (COX-2), macrophage colony-stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) using osteoblastic MC3T3-E1 cells and to examine the indirect effect of CF on osteoclast differentiation using RAW264.7 cells as osteoclast precursors. MC3T3-E1 cells were cultured with or without continuous CF (1.0 or 3.0 g/cm²) for 24 hr, and PGE₂ production was determined using ELISA. The expression of COX-2, M-CSF, RANKL, and OPG genes and proteins was determined using real-time PCR and ELISA, respectively. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of RAW 264.7 cells cultured for 10 days with conditioned medium from CF-treated MC3T3-E1 cells and soluble RANKL. As CF increased, PGE₂ production and the expression of COX-2, M-CSF, and RANKL increased, whereas OPG expression decreased. The number of TRAP-positive cells increased as CF increased. Celecoxib, a specific inhibitor of COX-2, blocked the stimulatory effect of CF on TRAP staining and the production of PGE₂, M-CSF, RANKL, and OPG. These results suggest that CF induces osteoclast differentiation by increasing M-CSF production and decreasing OPG production via PGE₂ in osteoblasts.     

KATP channel current increases in postinfarction remodeled cardiomyocytes

Adenosintriphosphate-sensitive potassium channels (K_ATP channels) are an important linkage between the metabolic state of a cell and electrophysiological membrane properties. In this study, K_ATP channels were studied in myocytes of normal and remodeled myocardium of the rat. Myocardial infarction was induced by ligature of the left anterior descending artery. Remodeled myocytes were obtained from the hypertrophied posterior left ventricular wall and interventricular septum 3 months after infarction. The current through K_ATP channels was measured in whole-cell and inside-out patches by using the patch-clamp technique. After myocardial infarction, the heart weight/body weight ratio was doubled and the myocytes were hypertrophied yielding a cell capacitance of 266±16 pF compared to 122±12 pF in control cells. The amount of Kir6.2 protein was indistinguishable in corresponding regions of control and remodeled hearts. The ATP sensitivity of K_ATP channels in remodeled cells was significantly lower than in control cells (half maximum block at 115 μmol/l ATP in remodeled and at 71 μmol/l ATP in control cells). The maximum I _KATP density induced by metabolic inhibition was higher in small remodeled (176±15 pA/pF) than in control cells (127±11 pA/pF), but was unchanged in large remodeled cells. Both, the higher I _KATP density and the lower sensitivity of the K_ATP channels to ATP suggest that remodeled cardiomyocytes develop an improved tolerance to ischemia by stabilizing the resting potential and decreasing excitability.     

JAK-STAT pathway modulates the roles of iNOS and COX-2 in the cytoprotection of early phase of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress

Our previous studies have demonstrated that preconditioning with hydrogen peroxide (H₂O₂) activated the JAK-STAT pathway that played an important role in the cytoprotection, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mediated the late phase of cytoprotection induced by high concentration of H₂O₂ after preconditioning. Here we sought to identify the downstream targets of the JAK-STAT axis that mediated H₂O₂ preconditioning and the expression of iNOS and COX-2 in the early phase of H₂O₂ preconditioning. It was shown that (1) Preconditioning with H₂O₂ at 100 μmol/L for 90 min in PC12 cells induced significant expression of iNOS and COX-2. (2) Pretreatment with the iNOS inhibitor AG (10 μmol/L) or the COX-2 inhibitor NS-398 (10 μmol/L) respectively 20 min before H₂O₂ preconditioning not only inhibits the increased expression of iNOS or COX-2 but also abrogates the protective effects of H₂O₂ preconditioning against apoptosis induced by oxidative stress. (3) Pretreatment with the JAK inhibitor AG-490 (10 μmol/L) 20 min before H₂O₂ preconditioning obviously inhibits the up-regulation of iNOS or COX-2 induced by H₂O₂ preconditioning. These results suggested that JAK-STAT pathway modulates the roles of iNOS and COX-2 in the cytoprotection of early phase of H₂O₂ preconditioning.     

Diadenosine polyphosphate-induced inhibition of cardiac KATP channels: Operative state-dependent regulation by a nucleoside diphosphate

It has been proposed that the regulatory action of mononucleotides, such as ATP and UDP, on cardiac ATP-sensitive K⁺ (K_ATP) channels is determined by the state of the channel. Recently, dinucleotides, such as diadenosine tetraphosphate (Ap₄A) and diadenosine pentaphosphate (Ap₅A), have been recognized as novel intracellular ligands of cardiac K_ATP channels. However, it is not known whether the state of K_ATP channels also determines the response of the channel to dinucleotides. Therefore, we examined the action of diadenosine polyphosphates on K_ATP channel activity during different operative channel states, using the inside-out patch clamp technique applied to patches excised from guinea-pig ventricular myocytes. Spontaneous openings of K_ATP channels (or operative condition 1) were inhibited by Ap₄A and Ap₅A. Addition of UDP, which on its own did not affect spontaneous openings of K_ATP channels, prevented Ap₄A and Ap₅A to inhibit spontaneous K_ATP channel activity. In contrast, following run-down of spontaneous channel activity, UDP induced K_ATP channel openings (or operative condition 2), but could no longer antagonize the inhibitory effect of Ap₄A and Ap₅A. We conclude that the inhibitory action of diadenosine polyphosphates on K_ATP channels can be blocked by UDP only when K_ATP channels are in operative condition 1, but not in operative condition 2. Thus, the operative condition of KATp channels determines the UDP-mediated regulation of the diadenosine polyphosphate-dependent inhibitory channel gating. This finding further suggests that the operative state of the cardiac K_ATP channel protein is a critical determinant of the regulatory action of an intracellular ligand.     

Expression of cyclooxygenase-1/-2, microsomal prostaglandin-E synthase-1 and E-prostanoid receptor 2 and regulation of inflammatory mediators by PGE2 in the amoeboid microglia in hypoxic postnatal rats and murine BV-2 cells

This study aimed to investigate the effect of hypoxia on the expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), microsomal prostaglandin-E synthase (mPGES-1), E-prostanoid receptor 2 (EP2) in microglia; and the roles of EP2-cyclic adenosine monophosphate (cAMP) signaling pathway in the prostaglandin E₂ (PGE₂) regulation of inflammatory mediators released by hypoxic BV-2 cells. Immunoexpression of COX-1, COX-2, mPGES-1 and EP2 was localized in the amoeboid microglial cells (AMC), a nascent brain macrophage in the developing brain, as confirmed by double labeling with OX-42 and lectin, specific markers of microglia. AMC emitted a more intense immunofluorescence in hypoxic rats when compared with the matching controls. In postnatal rats subjected to hypoxia, mRNA and protein expression levels of COX-1, COX-2 and mPGES-1 along with tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric-oxide synthase (iNOS) and PGE₂ product in the callosal tissue were significantly increased. The results were shared in the BV-2 cells except for COX-1 mRNA and protein whose levels remained unaltered. Interestingly, treatment with EP2 antagonist AH-6809 resulted in suppression of hypoxia induced EP2, IL-1β and iNOS mRNA and protein expression, TNF-α protein expression and intracellular cAMP level in BV-2 cells. It is suggested that PGE₂ may regulate above inflammatory mediators in the activated microglia via EP2-cAMP signaling pathway in hypoxic conditions.     

Anti-CD45 antibody enhances lipoxygenase pathway of human naïve mononuclear cells and cyclooxygenase pathway of neutrophils

Objective and Design: Anti-CD45 antibody exhibits multiple biological effects on human mononuclear cells (MNC) and polymorphonuclear neutrophils (PMN). We intended to determine whether anti-CD45 antibody could affect arachidonic acid metabolism and thereby, the interactions between human na?ve MNC and PMN. Materials and Methods: Human na?ve MNC and PMN were incubated with monoclonal anti-human CD45 IgG F(ab’)₂ antibody or non-specific IgG F(ab’)₂ for 30 min. The mRNA expression of cyclooxygenase type 1 (COX-1), type 2 (COX-2), 5-lipoxygenase (5-LOX) and leukotriene A₄ hydrolase (LTA₄ hydrolase) in both cells was detected by RT-PCR and quantified by densitometric determination. The presence of COX-1 and COX-2 molecules in the cells was detected by Western blot. The concentration of PGE₂ and LTB₄ in cultured supernatants was measured by EIA kits. Results: Anti-CD45 IgG F(ab’)₂ up-regulated LTA₄ hydrolase mRNA expression and LTB₄ production, but down-regulated COX-1 and COX-2 mRNA expression and PGE2 production, of na?ve MNC compared to non-specific IgG F(ab’)₂. In contrast, a reverse modulation by the specific antibody on PMN was observed including up-regulation of cyclooxygenase pathway and down-regulation of lipoxygenase pathway. Conclusions: A novel activity of anti-CD45 with reverse modulation on cyclooxygenase/lipoxygenase pathways was found such that the expression of COX-1 and COX-2 in PMN, and 5-LOX and LTA₄ hydrolase in MNC were enhanced. Received 4 May 2005; returned for revision 29 June 2005; returned for final revision 12 October 2005; accepted by M. Katori 16 November 2005     

Production of prostaglandin E₂ in response to infection with modified vaccinia Ankara virus

Prostaglandin E₂ (PGE₂) is an arachidonic acid (AA)-derived signaling molecule that can influence host immune responses to infection or vaccination. In this study, we investigated PGE₂ production in vitro by cells infected with the poxvirus vaccine strain, modified vaccinia Ankara virus (MVA). Human THP-1 cells, murine bone marrow-derived dendritic cells, and murine C3HA fibroblasts all accumulated PGE₂ to high levels in culture supernatants upon infection with MVA. We also demonstrated that MVA induced the release of AA from infected cells, and this was, most unusually, independent of host cytosolic phospholipase A₂ activity. The accumulation of AA and PGE₂ was dependent on viral gene expression, but independent of canonical NF-κB signaling via p65/RelA. The production of PGE₂ required host cyclooxygenase-2 (COX-2) activity, and COX-2 protein accumulated during MVA infection. The results of this study provide insight into a novel aspect of MVA biology that may affect the efficacy of MVA-based vaccines.     

Downregulation of inflammatory mediators and pro-inflammatory cytokines by alkaloids of jeevaneeya rasayana in adjuvant-induced arthritis

Jeevaneeya rasayana is an ayurvedic polyherbal formulation, with antirheumatic potential. The present study investigates the therapeutic efficacy of isolated total alkaloid fraction of Jeevaneeya Rasayana (AJR) in treating rheumatoid arthritis in a rat model of Adjuvant-induced arthritis (AIA). Paw swelling, inflammatory mediators such as cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), level of prostaglandin E₂ (PGE₂), expression of cytokines and serum nitric oxide (NO) level were analyzed in experimental rats after an experimental period of 21 days. Arthritic induction significantly increased paw edema, and up regulated the inflammatory mediators and cytokines. Administration of AJR significantly reversed the paw edema, reduced the level of PGE₂, serum NO and decreased the COX-2 activity in the paw tissue. AJR treatment also downregulated mRNA expression of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and MMP-9 in paw tissue. HPTLC analysis revealed the presence of 5 different alkaloid compounds in AJR. These findings suggest that the AJR have the therapeutic potential against adjuvant-induced arthritis.     

ATP4− and ATP·Mg inhibit the ATP-sensitive K+ channel of rat ventricular myocytes

K⁺ currents were recorded from ATP-sensitive channels in inside-out membrane patches excised from isolated rat ventricular myocytes. ATP-sensitive K⁺ channel inhibition could be evoked by ATP in the absence of magnesium where most ATP would be present as the free acid ATP^4–. Channel inhibition was enhanced when the same total concentration of ATP was applied in the presence of magnesium, where most ATP would be bound as ATP·Mg. Dose-response relationships for ATP-sensitive K⁺ channel inhibition evoked by ATP had a Hill coefficient of 2 andK _i of 17 and 30 M for ATP in the presence and absence of magnesium respectively. This was the obverse of the expected results if ATP^4– were to be the sole form of ATP to effect channel closure. ATP-sensitive K⁺ channel inhibition evoked by ATPS, AMP-PNP and AMP-PCP was also enhanced in the presence of magnesium. It is concluded that the ATP-sensitive K⁺ channel of rat ventricular myocytes binds and is closed by both the free-acid and divalent-cationbound forms of ATP.     

Exercise does not increase cyclooxygenase-2 myocardial levels in young or senescent hearts

Increased myocardial cyclooxygenase-2 (COX-2) activity is essential for late phase ischemic preconditioning (IPC). Currently unknown is whether cardioprotection elicited by exercise also involves elevated myocardial COX-2 activity. This investigation tested whether aerobic exercise elevates myocardial COX-2 protein content or enzyme activity in young and senescent male Fisher 344 rats assigned to sedentary or cardioprotective endurance exercise treatments (3 consecutive days of treadmill exercise, 60 min/day @~70% VO₂max). Assay of cardiac COX-2 protein content, catalytic activity, and inducible nitric oxide synthase (iNOS) protein content reveal that exercise did not alter COX-2 activity (PGE₂, p = 0.866; PGF1α, p = 0.796) or protein levels (p = 0.397) within young or senescent hearts. In contrast, myocardial iNOS, an up-stream mediator of COX-2 expression, was over-expressed by an average of 37% in aged hearts (p = 0.005), though iNOS was not influenced by exercise. Findings reveal exercise does not elevate cardiac COX-2 activity and suggests that mechanisms responsible for cardioprotection differ between IPC and aerobic exercise.     

Biophysical,pharmacological and developmental properties of ATP-sensitive K+ channels in cultured myotomal muscle cells from Xenopus embryos

Unlike mammalian muscle cells in culture, cultured myotomal muscle cells of Xenopus embryos express ATP-sensitive K⁺ (K_ATP) channels. The K_ATP channels are blocked by internal ATP (half-maximal inhibition K _0.5 = 16 M) and to a lesser extent by internal ADP, are voltage independent, have an inward rectification at positive potentials and are inhibited by glibenclamide (K _0.5 = 2 M). Surprisingly, these K_ATP channels are not sensitive to K⁺ channel openers such as cromakalim. Opening of these K_ATP channels does not occur under normal physiological conditions. It is elicited by metabolic exhaustion of the muscle cell and it precedes the development of an irreversible rigor state. Neither intracellular acidosis nor an increase of intracellular Ca²⁺ are involved in K_ATP channel opening. Different types of K⁺ channels are successively expressed after plating of myotomal muscle cells: (1) sustained delayed-rectifier K⁺ channels; (2) K_ATP channels; (3) inward-rectifier K⁺ channels; (4) transient delayed-rectifier K⁺ channels. The current density associated with K_ATP channels far exceeds that of voltage-dependent K⁺ channels. Innervation controls the expression of these K_ATP channels. Co-culture of muscle cells with neurons from the neural tube decreases the number of active K_ATP channels per patch. Similarly, in situ innervated submaxillaris muscle of tadpoles at stage 50–55 has a very low density of K_ATP channels.     

Comparative Study of Normal and Rheumatoid Arthritis Fibroblast-Like Synoviocytes Proliferation under Cyclic Mechanical Stretch: Role of Prostaglandin E2

Fibroblast-like synoviocytes (FLSs) are one of the main contributors of prostaglandin E₂ (PGE₂) in the hyperplastic synovium of rheumatoid arthritis (RA) patients. cyclooxygenase-2 (COX-2)/PGE₂ pathway is involved in the proliferation of several cell types. We have previously shown that mechanical stretch affects COX-2 and PGE₂ production in human RA FLSs; however, its role in cell proliferation remains to be elucidated. In this study, a comparison is drawn between human RA and normal FLSs to understand the role of mechanical stretch and PGE₂ on the proliferation of FLSs. The results showed that physiological level (6%, 1 Hz) of cyclic mechanical stretch significantly decreased the proliferation of RA FLSs but not normal FLSs, while the induction of apoptosis was not observed by stretch in either RA or normal FLSs. IL-1β (5 ng/ml)-induced COX-2/PGE₂ levels are downregulated by stretch in RA FLSs only. Further investigation showed that high concentration (100 and 500 ng/ml) of PGE₂ significantly induced cell proliferation only in RA FLSs, and this induction failed to be suppressed by stretch. In conclusion, this study demonstrated that elevated levels of PGE₂ in the synovial cavity are involved in the proliferation of RA FLSs, and cyclic mechanical stretch regulates the RA synovial hyperplasia.