Melatonin suppresses vasospastic effect of hydrogen peroxide in human umbilical artery: Relation to calcium influx

ABSTRACT: We evaluated the hydroxyl radical scavenging effect of melatonin on the vasospastic action induced by hydrogen peroxide in human umbilical artery. Helical sections were made of umbilical arteries obtained from healthy pregnant women who were delivered between 37 and 39 weeks of gestation. Changes in maximal potassium chloride (KC1, 10^?2 M)-induced tension were measured in umbilical artery segments with intact endothelium. Segments were treated with H₂O₂ (10^?9 M to 10^?7 M) only, or were pretreated with an H₂O₂ scavenger (catalase, 2,000 IU), a hydroxyl radical scavenger (mannitol, 10^?2 M), or melatonin (10^?8 M to 10^?6 M). The effect of H₂O₂ on the response of the segments of umbilical artery to external calcium was determined. Changes in KCl-induced contraction were also determined in segments pretreated with an inhibition of intracellular calcium release (ryanodine, 10^?4 M) prior to exposure to H₂O₂. Pretreating the segments of umbilical arteries with H₂O₂ (10^?8 M, 10^?7 M) significantly potentiated the maximal contraction induced by KC1 (P < 0.0001, P < 0.03, respectively). Pretreatment with either catalase or mannitol significantly reduced the vasospastic effect of H₂O₂ (10^?8 M) (P < 0.0001, P < 0.0001, respectively). Melatonin also significantly reduced the vasospastic effect of H₂O₂ (10^?8 M), in a concentration-dependent manner (P<0.0001). H₂O₂ (10^?8 M) significantly increased the contractile response to external calcium. Melatonin pretreatment significantly suppressed the contractile response to external calcium. Treatment with ryanodine prior to exposure to H₂O₂ did not affect KCl-induced contraction. Results suggest that H₂O₂ potentiates the KCl-induced maximal contraction of the human umbilical artery, perhaps by increasing calcium influx via activation of the voltage-dependent calcium channel. Melatonin significantly suppresses the vasospastic effect of H₂O₂, probably due to its scavenging of the hydroxyl radical.     

Attenuation by melatonin of human umbilical arterial vasoconstriction induced by lysophosphatidylcholine

We evaluated the antioxidant property of melatonin as related to the vasospastic effect of lysophosphatidylcholine (LPC), a component of oxidized lipoprotein, on the human umbilical artery. Helical sections of umbilical arteries were obtained from healthy pregnant women who were delivered between 37 and 39 wk of gestation. Changes in maximal tension induced by KCl were measured in arterial sections having intact endothelium. Sections were treated with LPC alone (15 or 30 microM), or were pretreated either with a hydrogen peroxide (H2O2) scavenger (catalase, 1,200 U/mL), a hydroxyl radical scavenger (mannitol, 30 mM), a nitric oxide (NO) synthesis inhibitor (L-N(G)-monomethyl arginine, LNMA, 2 x 10(-4) M) or melatonin (1 or 10 microM). The effect of LPC (30 microM) on the vasorelaxation induced by 5-hydroxytryptamine (5-HT) was also determined, with or without melatonin pretreatment (10 microM). LPC potentiated vascular tension in a concentration-dependent manner. Pretreatment with LNMA significantly suppressed this vasospastic effect of LPC. Pretreatment with catalase or mannitol significantly reduced the vasospastic effect of LPC. Melatonin significantly lessened the vasospastic effect of LPC in a concentration-dependent manner. Pretreatment with LPC significantly inhibited the relaxation induced by 5-HT. Treatment with melatonin prior to LPC exposure significantly restored the relaxation induced by 5-HT. Results suggest that LPC potentiates vascular tension in human umbilical artery, perhaps by suppressing the endothelial synthesis of NO. Melatonin significantly suppressed the vasospastic effect of LPC. This agent probably scavenges the hydroxyl radicals arising from LPC.     

Melatonin suppresses lysophosphatidylcholine enhancement of serotonin-induced vasoconstriction in the human umbilical artery

We evaluated the antioxidant property of melatonin as related to the vasospastic effect of lysophosphatidylcholine (LPC), a component of oxidized lipoprotein, on the human umbilical artery. Helical sections of umbilical arteries with intact endothelium were obtained at elective cesarean delivery between 37 and 39 weeks of gestation. Changes in 5-hydroxytryptamine (5-HT)-induced vasoconstriction were measured. Arterial sections were treated with LPC alone (15 or 30 microM) or pretreated with an hydroxyl radical (.OH) scavenger (mannitol), a cyclooxygenase inhibitor (indomethacin, 20 microM), nitric oxide (NO) synthesis inhibitor (L-N(G)-monomethylarginine, LNMA, 2 x 10(-4) M), or melatonin (1 or 10 microM). LPC potentiated 5-HT-induced contraction in a concentration-dependent manner. Pretreatment with mannitol significantly suppressed the vasospastic effect of LPC. LNMA augmented the vasospastic effect of LPC, but indomethacin did not. Melatonin significantly suppressed the vasospastic effect of LPC in a concentration-dependent manner. Considering a previous finding that .OH and oxidized low-density lipoprotein decrease NO production in the human umbilical artery, the vasospastic effect of LPC appear to involve suppression of endothelial NO synthesis. Melatonin significantly suppresses the vasospastic effect of LPC, probably by scavenging .OH arising from LPC.     

Melatonin suppresses homocysteine enhancement of serotonin-induced vasoconstriction in the human umbilical artery

Hyperhomocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (Hcy)-induced endothelial dysfunction. Herein, we evaluated the antioxidant property of melatonin as related to the vasospastic effect of Hcy on the human umbilical artery. Helical strips of human umbilical arteries with intact endothelium were obtained at elective Caesarean delivery between 37 and 39 wks of gestation. Changes in 5-hydroxytryptamine (5-HT)-induced vasoconstriction were measured. Arterial strips were treated with FeSO4 (10 microM) and Hcy (10 or 100 microM) or pre-treated with a hydroxyl radical (.OH) scavenger (mannitol, 20 mM), a cyclooxygenase inhibitor (indomethacin, 20 microM), nitric oxide (NO) synthesis inhibitor (L-NG-monomethylarginine, LNMA, 200 microM), or melatonin (1 or 10 microM). Hcy potentiated 5-HT-induced constriction in a concentration-dependent manner. Pre-treatment with mannitol significantly suppressed the vasospastic effect of Hcy. LNMA augmented the vasospastic effect of Hcy, but indomethacin did not. Melatonin significantly suppressed the vasospastic effect of Hcy in a concentration-dependent manner. These findings suggest that Hcy potentiates 5-HT-induced vasoconstriction in the human umbilical artery, possibly by suppressing bioavailable NO. Melatonin protects against the vasospastic effect of Hcy, most likely by scavenging.OH arising from Hcy autooxidation.     

Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H₂O₂) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H₂O₂ at a sublethal concentration of 200 μm , human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H₂O₂ exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H₂O₂ exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H₂O₂‐induced premature senescence. We also found that melatonin attenuated the H₂O₂‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16^INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.     

Melatonin promotes human oocyte maturation and early embryo development by enhancing clathrin‐mediated endocytosis

Embryo development potential and reproductive clinical outcomes are all deeply rooted in oocyte maturation. Melatonin has been reported to promote oocyte maturation as an antioxidant in nonprimate species. Its antioxidative functions also help reduce plasma membrane rigidity, which facilitates clathrin‐mediated endocytosis (CME). Whether melatonin has effects on human oocyte maturation by regulating CME is worthy of exploration. In this study, we found that the optimal melatonin concentration for human oocyte maturation was 10^?11 M, and the maturation rate of this group was 71.9% (P = .03). The metaphase II (MII) stage oocytes obtained by in vitro maturation with 10^?11 M melatonin had a significantly higher fertilization rate (81.4% vs 61.4%, respectively, P = .017) and blastocyst rate (32.2% vs 15.8%, respectively, P = .039) compared to controls. During maturation, antioxidative melatonin greatly enhanced CME and decreased intra‐oocyte cAMP level. The former was evidenced by the increasing numbers of coated pits and vesicles, and the upregulated expression of two major CME markers—clathrin and adaptor protein‐2 (AP2). CME inhibitor dynasore increased intra‐oocyte cAMP level and blocked oocyte maturation, and melatonin could partly rescue oocyte maturation and significantly elevate the expression of clathrin and AP2 in the presence of dynasore. Therefore, we conclude that melatonin could promote human oocyte maturation and early embryo development through enhancing CME.     

Exogenous melatonin improves Malus resistance to Marssonina apple blotch

We examined whether exogenously applied melatonin could improve resistance to Marssonina apple blotch (Diplocarpon mali) by apple [Malus prunifolia (Willd.) Borkh. cv. Donghongguo]. This serious disease leads to premature defoliation in the main regions of apple production. When plants were pretreated with melatonin, resistance was increased in the leaves. We investigated the potential roles for melatonin in modulating levels of hydrogen peroxide (H₂O₂), as well the activities of antioxidant enzymes and pathogenesis‐related proteins during these plant–pathogen interactions. Pretreatment enabled plants to maintain intracellular H₂O₂ concentrations at steady‐state levels and enhance the activities of plant defence‐related enzymes, possibly improving disease resistance. Because melatonin is safe and beneficial to animals and humans, exogenous pretreatment might represent a promising cultivation strategy to protect plants against this pathogen infection.     

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels

Summary Hydrogen peroxide (H₂O₂) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H₂O₂ was markedly attenuated by 10μM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100nM charybdotoxin, an inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels. A combination of the ODQ and charybdotoxin abolished the H₂O₂-induced relaxation. Pretreatment with 25 μM of an Rp stereoisomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), 20μM glibenclamide, or 1mM 4-aminopyridine did not affect the vascular response to H₂O₂. The presence of catalase at 1000U/ml significantly attenuated the H₂O₂-induced relaxation. Exposure of cultured smooth muscle cells to H₂O₂ activated K_Ca channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of K_Ca channels. Rp-cAMPS did not inhibit the H₂O₂-induced activation of K_Ca channels. The activation of K_Ca channels by H₂O₂ was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H₂O₂ activated K_Ca channels in a concentration-dependent manner. In inside-out patches, H₂O₂ significantly activated K_Ca channels through a process independent of cyclic guanosine 3′,5′-monophosphate (cGMP). In conclusion, H₂O₂ elicits vascular relaxation due to activation of K_Ca channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.     

Melatonin exists in porcine follicular fluid and improves in vitro maturation and parthenogenetic development of porcine oocytes

Abstract: This study focused on the effect of melatonin on in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Melatonin was measured in porcine follicular fluid of follicles of different sizes in the same ovary. Melatonin exists in follicular fluid, and the concentration is approximately 10^?11 m . Its concentration decreased as the diameter of follicle increased, which suggests an effect of melatonin on oocyte maturation. Therefore, immature oocytes were cultured in vitro in maturation medium supplemented with melatonin (10^?11, 10^?9, 10^?7, 10^?5 and 10^?3 m ) or without melatonin. The oocytes at maturation stage were collected and activated. The parthenogenetic embryos were cultured and observed in medium supplemented with or without melatonin. Fresh immature oocytes without melatonin treatment were used as control. When only maturation medium was supplemented with 10^?9 m melatonin, the cleavage rate, blastocyst rate and the cell number of blastocyst (70 ± 4.5%, 28 ± 2.4% and 50 ± 6.5%) were significantly higher (P < 0.05) than that of controls; when only culture medium was supplemented with melatonin, the highest cleavage rate, blastocyst rate and the cell number of blastocyst was observed at 10^?7 m melatonin, which were significantly higher than that of controls (P < 0.05). The best results (cleavage rates 79 ± 8.4%, blastocyst rates 35 ± 6.7%) were obtained when both the maturation and culture medium were supplemented with 10^?9 m melatonin respectively (P < 0.05). In conclusion, exogenous melatonin at the proper concentration may improve the in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Further research is needed to identify the effect of melatonin on in vitro and in vivo oocyte maturation and embryo development in porcine.     

Phytomelatonin receptor PMTR1‐mediated signaling regulates stomatal closure in Arabidopsis thaliana

Melatonin has been detected in plants in 1995; however, the function and signaling pathway of this putative phytohormone are largely undetermined due to a lack of knowledge about its receptor. Here, we discovered the first phytomelatonin receptor (CAND2/PMTR1) in Arabidopsis thaliana and found that melatonin governs the receptor‐dependent stomatal closure. The application of melatonin induced stomatal closure through the heterotrimeric G protein α subunit‐regulated H₂O₂ and Ca²⁺ signals. The Arabidopsis mutant lines lacking AtCand2 that encodes a candidate G protein‐coupled receptor were insensitive to melatonin‐induced stomatal closure. Accordingly, the melatonin‐induced H₂O₂ production and Ca²⁺ influx were completely abolished in cand2. CAND2 is a membrane protein that interacts with GPA1 and the expression of AtCand2 was tightly regulated by melatonin in various organs and guard cells. CAND2 showed saturable and specific ¹²⁵I‐melatonin binding, with apparent K_d (dissociation constant) of 0.73 ± 0.10 nmol/L (r² = .99), demonstrating this protein is a phytomelatonin receptor (PMTR1). Our results suggest that the phytomelatonin regulation of stomatal closure is dependent on its receptor CAND2/PMTR1‐mediated H₂O₂ and Ca²⁺signaling transduction cascade.     

Melatonin inhibits vasospastic action of oxidized low-density lipoprotein in human umbilical arteries

We evaluated the antioxidant property of melatonin in countering the vasospastic effect of oxidized low-density lipoprotein (ox-LDL), which has been reported to be the most important risk factor for atherosclerosis and also may be linked to preeclampsia. Helical sections of umbilical arteries were obtained from human placentas at elective cesarean deliveries between 37 and 39 weeks of gestation. Changes in maximal tension induced by potassium chloride were measured in arterial sections with intact endothelium. ox-LDL (200 or 300 microg protein/mL) increased vascular tension by 15.6 +/- 2.3 or 31.9 +/- 4.0%, respectively. In contrast, native LDL only slightly increased vascular tension (2.7 +/- 1.0% for 200 microg protein/mL and 6.0 +/- 1.7% for 300 microg protein/mL). Pretreatment with L-N(G)-monomethyl-arginine (2 x 10(-4) M) significantly reduced the vasospastic effect of ox-LDL, as did pretreatment with mannitol (30 mM). Melatonin (10 microM) significantly reduced the vasospastic effect of ox-LDL. These findings suggest that ox-LDL potentiates vascular tension in the human umbilical artery, possibly by suppressing endothelial synthesis of nitric oxide. Melatonin significantly suppressed the vasospastic effect of ox-LDL, probably because it scavenges hydroxyl radical arising from ox-LDL.     

Melatonin improves the reprogramming efficiency of murine‐induced pluripotent stem cells using a secondary inducible system

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all‐iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10^?6, 10^?7, 10^?8, 10^?9, or 10^?10 m ) or with vitamin C (50 μg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10^?8 (0.81 ± 0.04%), and 10^?9 m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (P < 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT₂ was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT₁ and MT₂ receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis‐related genes p53 and p21 was lower in the group treated with 10^?9 m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53‐mediated apoptotic pathway.     

Melatonin enhances the occurrence of autophagy induced by oxidative stress in Arabidopsis seedlings

The beneficial effect that melatonin has against mitochondrial dysfunctioning seems to be linked to mitophagy. Roles for melatonin have been demonstrated in promoting health and preventing disease, as well as activating the process of autophagy in general. However, no reports have been made about how the application of melatonin regulates that process when plants are exposed to oxidative stress. We investigated the influence of different concentrations of melatonin (0.0, 0.5, 5.0, 10.0, or 50.0 μm ) on autophagy under methyl viologen (MV)‐induced oxidative stress. Arabidopsis seedlings that were pretreated with 5 or 10 μm melatonin underwent relatively strong induction of autophagy, as evidenced by the number of monodansylcadaverine (MDC)‐stained autophagosomes in root samples. Pretreatment with 10 μm melatonin also alleviated MV‐induced photo‐oxidation damage and significantly reduced the accumulation of oxidized proteins. Those responses might have been due to the strong upregulation of genes that involved in AtATG8‐PE conjugation pathway, which enhanced the capacity for autophagy. Histochemical staining revealed that both and H₂O₂ were highly accumulated upon MV exposure, although the response did not differ significantly between control and melatonin‐pretreated seedlings. By contrast, exogenous melatonin upregulated the expression of two genes for H₂O₂‐scavenging enzymes, that is, AtAPX1 and AtCATs. The activation of autophagy by melatonin without an alteration in ROS production may be part of a survival mechanism that is enhanced by melatonin after cellular damage. Therefore, it represents a second level of defense to remove damaged proteins when antioxidant activities are compromised.     

Melatonin and its derivatives cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine and 6-methoxymelatonin reduce oxidative DNA damage induced by Fenton reagents

Abstract: Free radicals are generated in vivo and they oxidatively damage DNA because of their high reactivities. In the last several years, hundreds of publications have confirmed that melatonin is a potent endogenous free radical scavenger. Some of the metabolites produced as a result of these scavenging actions have been identified using pure chemical systems. This is the case with both N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK), identified as a product of the scavenging reaction of H₂O₂ by melatonin, and cyclic 3‐hydroxymelatonin (C‐3‐OHM) which results when melatonin detoxifies two hydroxyl radicals (?OH). In the present in vitro study, we investigated the potential of two different derivatives of melatonin to scavenger free radicals. One of these derivatives is C‐3‐OHM, while the other is 6‐methoxymelatonin (6‐MthM). We also examined the effect of two solvents, i.e., methanol and acetonitrile, in this model system. As an endpoint, using high‐performance liquid chromatography we measured the formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OH‐dG) in purified calf thymus DNA treated with the Fenton reagents, chromium(III) [Cr(III)] plus H₂O₂, in the presence and in the absence of these molecules. The 8‐OH‐dG is considered a biomarker of oxidative DNA damage. Increasing concentrations of Cr(III) (as CrCl₃) and H₂O₂ was earlier found to induce progressively greater levels of 8‐OH‐dG in isolated calf thymus DNA because of the generation of ?OH via the Fenton‐type reaction. We found that C‐3‐OHM reduces ?OH‐mediated damage in a dose‐dependent manner, with an IC₅₀ = 5.0 ± 0.2 μm ; melatonin has an IC₅₀ = 3.6 ± 0.1 μm . These values differ statistically significantly with P < 0.05. In these studies, AFMK had an IC₅₀ = 17.8 ± 0.7 μm (P < 0.01). The 6‐MthM also reduced DNA damage in a dose‐dependent manner, with an IC₅₀ = 4.2 ± 0.2 μm ; this value does not differ from the ICs for melatonin and C‐3‐OHM. We propose a hypothetical reaction pathway in which a mole of C‐3‐OHM scavenges 2 mol of ?OH yielding AFMK as a final product. As AFMK is also a free radical scavenger, the action of melatonin as a free radical scavenger is a sequence of scavenging reactions in which the products are themselves scavengers, resulting in a cascade of protective reactions.     

Roles of Ca2+ and protein kinase C in regulation of prostaglandin E2 release by cultured rabbit gastric epithelial cells

Prostaglandin (PG) has been reported to be one of the important protective factors in the gastric mucosa. However the mechanism of the regulation of endogenous PG production has not been well studied. We investigated the possible roles of Ca²⁺, cAMP, and protein kinase C (PKC) in the regulation of PGE₂ release from cultured rabbit gastric mucosal cells. PGE₂ was measured by radioimmunoassay. A23187 (Ca²⁺ ionophore) at 2×10^?6 M significantly increased PGE₂ release. Deprivation of Ca²⁺ from the medium blocked the A23187-induced increase of PGE₂. TMB-8 (a putative inhibitor of Ca²⁺ release from intracellular stores) did not have any significant effects on the increase of PGE₂-induced by A23187. Thus, A23187 increased PGE₂ through the influx of extracellular Ca²⁺. W7 or compound 48/80 (calmodulin inhibitors) did not alter the response of PGE₂ caused by A23187. Exogenous administration of cAMP, forskolin (an activator of adenylate cyclase), or 2-chloroadenosine (a possible activator of adenylate cyclase through adenosine A₂ receptor) had neither significant effects on PGE₂ release nor an effect on A23187-induced increase of PGE₂ release. 12-O-tetradecanoylphorbol 13-acetate (TPA, an activator of PKC) significantly stimulated PGE₂ release in a dose-dependent fashion, whereas another phorbol ester with no biological activity did not. A23187 at 0.8×10^?6 M, but not cAMP, potentiated the TPA-induced increase of PGE₂. Mepacrine (a phospholipase A₂ inhibitor) reduced the A23187-and TPA-induced increase of PGE₂. These results suggest that Ca²⁺ and protein kinase C may play important roles in the regulation of PGE₂ release by cultured rabbit gastric cells.     

Long noncoding RNA H19 mediates melatonin inhibition of premature senescence of c‐kit+ cardiac progenitor cells by promoting miR‐675

Melatonin, a hormone secreted by the pineal gland, possesses multiple biological activities such as antitumor, antioxidant, and anti‐ischemia. C‐kit⁺ cardiac progenitor cells (CPCs) have emerged as a promising tool for the treatment of heart diseases. However, the senescence of CPCs due to pathological stimuli leads to the decline of CPCs' functions and regenerative potential. This study was conducted to demonstrate whether melatonin antagonizes the senescence of CPCs in response to oxidative stress. Here, we found that the melatonin treatment markedly inhibited the senescent characteristics of CPCs after exposed to sublethal concentration of H₂O₂, including the increase in senescence‐associated β‐galactosidase (SA‐β‐gal)‐positive CPCs, senescence‐associated heterochromatin loci (SAHF), secretory IL‐6 level, and the upregulation of p53 and p21 proteins. Senescence‐associated proliferation reduction was also attenuated by melatonin in CPCs. Luzindole, the melatonin membrane receptor blocker, may block the melatonin‐mediated suppression of premature senescence in CPCs. Interestingly, we found that long noncoding RNA H19 and its derived miR‐675 were downregulated by H₂O₂ in CPCs, but melatonin treatment could counter this alteration. Furthermore, knockdown of H19 or miR‐675 blocked antisenescence actions of melatonin on H₂O₂‐treated CPCs. It was further verified that H19‐derived miR‐675 targeted at the 3′UTR of USP10, which resulted in the downregulation of p53 and p21 proteins. In summary, melatonin antagonized premature senescence of CPCs via H19/miR‐675/USP10 pathway, which provides new insights into pharmacological actions and potential applications of melatonin on the senescence of CPCs.     

Hypoxia Increases Superoxide Anion Production from Bovine Coronary Microvessels,But Not Cardiac Myocytes,via Increased Xanthine Oxidase

Objective: To determine if 30 min of hypoxia (Po² 8–10 Torr) affects basal, mitochondrial, or xanthine oxidase-derived lucigenin-detectable superoxide anion (O²_.-) production by intact isolated bovine coronary microvessels and myocytes. Methods: O₂^.- was quantitated by lucigenin-elicited chemiluminescence. Antimycin A (10 μM) and hypoxanthine (0.1 mM) were employed to increase O₂^.- from mitochondria and xanthine oxidase, respectively. Results: Chemiluminescence from microvessels and myocytes was enhanced (～ twofold, P < 0.05, n = 8–10) by inhibition of Cu,Zn-SOD via pretreatment with diethyldithiocarbamate (10 mM, 30 min) and was decreased (P < 0.05, n = 8–10) by an intracellular scavenger of O₂^.- (10 mM Tiron), but not by added SOD (3 μM, n = 8–10). In the presence of SOD inhibition, hypoxia produced a hypoxanthine-dependent (n = 8–10) twofold increase in chemiluminescence (P < 0.05, n = 10) in microvessels but not in myocytes. Other combinations of hypoxia, antimycin, or hypoxanthine did not significantly alter chemiluminescence. Conclusions: Lucigenin appears to detect a basal intracellular source of O^2.- in both microvessels and myocytes that is not derived from mitochondria or xanthine oxidase. Exposure to hypoxia does not appreciably increase basal O₂^.- in vessels or myocytes, but if exogenous hypoxanthine is supplied, microvessels show an increase in O₂^.- production presumably derived from xanthine oxidase.     

Lipoprotein lipase gene variants and progression of nephropathy in hypercholesterolaemic patients with type 2 diabetes

Objective. Recent prospective studies have identified hyperlipidaemia as an independent determinant of diabetic nephropathy. Lipoprotein lipase (LPL) is a key enzyme in the postprandial processing of triglycerides and VLDL. Among a number of common sequence variants of the LPL, HindIII has been associated with coronary heart disease and, more recently, with microalbuminuria in type 2 diabetes. We evaluated the progression of renal disease in hypercholesterolaemic type 2 diabetic patients in relation to this polymorphism. Design and subjects. We followed up for 4 years 65 consecutively enrolled microalbuminuric patients with type 2 diabetes; of whom 28 had hypercholesterolaemia (6.62 ± 0.9 mmol L^?1, group A) and 37 were normocholesterolaemic (4.68 ± 0.5 mmol L^?1, group B). Main outcome measures. After performing the genetic analyses, albumin excretion rate (AER) and estimated glomerular filtration rate (GFR), calculated by the simplified equation of the MDRD Study Group, were repeated every year. Results. In group A, AER increased more (?AER: 11 [38] vs. 4 [18] μg min^?1 per year in group B, P < 0.0001) while GFR declined faster (?3.5 ± 2.1 vs. ?2.0 ± 1.4 mL min^?1 per year, P < 0.02). Patients homozygous for the allele + of HindIII showed a significantly faster decline of GFR and a higher increase of AER (both P = 0.0001) even after adjustment for cholesterol levels and anthropometric variables. Conclusions. In hypercholesterolaemic type 2 diabetic patients with microalbuminuria, the renal disease has an accelerated course, particularly in those carrying the H₊/H₊ genotype of the HindIII polymorphism at the LPL locus.