A non-retinoid antagonist of retinol-binding protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD.

Cyclophilin D disruption attenuates lipopolysaccharide-induced inflammatory response in primary mouse macrophages.

According to recent results, various mitochondrial processes can actively regulate the immune response. In the present report, we studied whether mitochondrial permeability transition (mPT) has such a role. To this end, we compared bacterial lipopolysaccharide (LPS)-induced inflammatory response in cyclophilin D (CypD) knock-out and wild-type mouse resident peritoneal macrophages. CypD is a regulator of mPT; therefore, mPT is damaged in CypD(-/-) cells. We chose this genetic modification-based model because the mPT inhibitor cyclosporine A regulates inflammatory processes by several pathways unrelated to the mitochondria. The LPS increased mitochondrial depolarisation, cellular and mitochondrial reactive oxygen species production, nuclear factor-κB activation, and nitrite- and tumour necrosis factor α accumulation in wild-type cells, but these changes were diminished or absent in the CypD-deficient macrophages. Additionally, LPS enhanced Akt phosphorylation/activation as well as FOXO1 and FOXO3a phosphorylation/inactivation both in wild-type and CypD(-/-) cells. However, Akt and FOXO phosphorylation was significantly more pronounced in CypD-deficient compared to wild-type macrophages. These results provide the first pieces of experimental evidence for the functional regulatory role of mPT in the LPS-induced early inflammatory response of macrophages.

Quercetin increases the efficacy of glioblastoma treatment compared to standard chemoradiotherapy by the suppression of PI-3-kinase-Akt pathway.

The goal of the present study was to compare the efficacy of treatment with irradiation (IR), temozolomide, and quercetin, alone, or in combinations, on 2 glioblastoma cell lines, DBTRG-05 and U-251. Cell viability assay, flow cytometry analysis, colony formation assay, and Western blot analysis were used to compare the effects of treatment on the 2 cell lines. The greatest reduction in cell viability and colony formation was observed when cells were treated with a combination of the agents including quercetin. The treatment of cells with the combination of IR and quercetin was equal to the efficiency of the combination of IR and temozolomide in decreasing cell viability as well as colony formation. Quercetin alone, or in combination with IR, increased the cleavage of caspase-3 and PARP-1 showing an activated apoptosis and significantly reduced the level of phospho-Akt. Moreover, these treatments increased the levels of phospho-ERK, phospho-JNK, phospho-p38, and phospho-RAF1. Our data indicate that the supplementation of standard therapy with quercetin increases efficacy of treatment of experimental glioblastoma through synergism in the induction of apoptosis via the cleavage of caspase-3 and PARP-1 and by the suppression of the actitivation of Akt pathway.

Protective effect of the poly(ADP-ribose) polymerase inhibitor PJ34 on mitochondrial depolarization-mediated cell death in hepatocellular carcinoma cells involves attenuation of c-Jun N-terminal kinase-2 and protein kinase B/Akt activation.

BACKGROUND: 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11) was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose)-polymerase (PARP), protein kinase B/Akt and mitogen activated protein kinase (MAPK) activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. RESULTS: We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2), and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. CONCLUSIONS: These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

Facilitation of mitochondrial outer and inner membrane permeabilization and cell death in oxidative stress by a novel Bcl-2 homology 3 domain protein.

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x(L), which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.

Identification of Transthyretin Tetramer Kinetic Stabilizers That Are Capable of Inhibiting the Retinol-Dependent Retinol Binding Protein 4-Transthyretin Interaction: Potential Novel Therapeutics for Macular Degeneration, Transthyretin Amyloidosis, and Their Common Age-Related Comorbidities.

Dissociation of transthyretin (TTR) tetramers may lead to misfolding and aggregation of proamyloidogenic monomers, which underlies TTR amyloidosis (ATTR) pathophysiology. ATTR is a progressive disease resulting from the deposition of toxic fibrils in tissues that predominantly presents clinically as amyloid cardiomyopathy and peripheral polyneuropathy. Ligands that bind to and kinetically stabilize TTR tetramers prohibit their dissociation and may prevent ATTR onset. Drawing from clinically investigated AG10, we designed a constrained congener (14) that exhibits excellent TTR tetramer binding potency, prevents TTR aggregation in a gel-based assay, and possesses desirable pharmacokinetics in mice. Additionally, 14 significantly lowers murine serum retinol binding protein 4 (RBP4) levels despite a lack of binding at that protein's all-trans-retinol site. We hypothesize that kinetic stabilization of TTR tetramers via 14 is allosterically hindering all-trans-retinol-dependent RBP4-TTR tertiary complex formation and that the compound could present ancillary therapeutic utility for indications treated with RBP4 antagonists, such as macular degeneration.

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells.

BACKGROUND INFORMATION: PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). RESULTS: SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. CONCLUSION: Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

Novel neuroprotective strategies in ischemic retinal lesions.

Retinal ischemia can be effectively modeled by permanent bilateral common carotid artery occlusion, which leads to chronic hypoperfusion-induced degeneration in the entire rat retina. The complex pathways leading to retinal cell death offer a complex approach of neuroprotective strategies. In the present review we summarize recent findings with different neuroprotective candidate molecules. We describe the protective effects of intravitreal treatment with: (i) urocortin 2; (ii) a mitochondrial ATP-sensitive K(+) channel opener, diazoxide; (iii) a neurotrophic factor, pituitary adenylate cyclase activating polypeptide; and (iv) a novel poly(ADP-ribose) polymerase inhibitor (HO3089). The retinoprotective effects are demonstrated with morphological description and effects on apoptotic pathways using molecular biological techniques.

Comparative pharmacokinetics and pharmacodynamics of the advanced Retinol-Binding Protein 4 antagonist in dog and cynomolgus monkey.

Accumulation of lipofuscin bisretinoids in the retina contributes to pathogenesis of macular degeneration. Retinol-Binding Protein 4 (RBP4) antagonists reduce serum retinol concentrations thus partially reducing retinol delivery to the retina which decreases bisretinoid synthesis. BPN-14136 is a novel RBP4 antagonist with good in vitro potency and selectivity and optimal rodent pharmacokinetic (PK) and pharmacodynamic (PD) characteristics. To select a non-rodent species for regulatory toxicology studies, we conducted PK and PD evaluation of BPN-14136 in dogs and non-human primates (NHP). PK properties were determined following oral and intravenous administration of BPN-14136 in beagle dogs and cynomolgus monkeys. Dynamics of plasma RBP4 reduction in response to compound administration was used as a PD marker. BPN-14136 exhibited favorable PK profile in both species. Dose-normalized exposure was significantly higher in NHP than in dog. Baseline concentrations of RBP4 were considerably lower in dog than in NHP, reflecting the atypical reliance of canids on non-RBP4 mechanisms of retinoid trafficking. Oral administration of BPN-14136 to NHP induced a strong 99% serum RBP4 reduction. Dynamics of RBP4 lowering in both species correlated with compound exposure. Despite adequate PK and PD characteristics of BPN-14136 in dog, reliance of canids on non-RBP4 mechanisms of retinoid trafficking precludes evaluation of on-target toxicities for RBP4 antagonists in this species. Strong RBP4 lowering combined with good PK attributes and high BPN-14136 exposure achieved in NHP, along with the biology of retinoid trafficking that is similar to that of humans, support the choice of NHP as a non-rodent safety species.

Discovery of Bispecific Antagonists of Retinol Binding Protein 4 That Stabilize Transthyretin Tetramers: Scaffolding Hopping, Optimization, and Preclinical Pharmacological Evaluation as a Potential Therapy for Two Common Age-Related Comorbidities.

Accumulation of cytotoxic lipofuscin bisretinoids may contribute to atrophic age-related macular degeneration (AMD) pathogenesis. Retinal bisretinoid synthesis depends on the influx of serum all-trans-retinol (1) delivered via a tertiary retinol binding protein 4 (RBP4)-transthyretin (TTR)-retinol complex. We previously identified selective RBP4 antagonists that dissociate circulating RBP4-TTR-retinol complexes, reduce serum RBP4 levels, and inhibit bisretinoid synthesis in models of enhanced retinal lipofuscinogenesis. However, the release of TTR by selective RBP4 antagonists may be associated with TTR tetramer destabilization and, potentially, TTR amyloid formation. We describe herein the identification of bispecific RBP4 antagonist-TTR tetramer kinetic stabilizers. Standout analogue (±)-44 possesses suitable potency for both targets, significantly lowers mouse plasma RBP4 levels, and prevents TTR aggregation in a gel-based assay. This new class of bispecific compounds may be especially important as a therapy for dry AMD patients who have another common age-related comorbidity, senile systemic amyloidosis, a nongenetic disease associated with wild-type TTR misfolding.

Influence of PACAP on oxidative stress and tissue injury following small-bowel autotransplantation.

Tissue injury caused by cold preservation and reperfusion remains an unsolved problem during small-bowel transplantation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present and plays a central role in the intestinal physiology. This study investigated effect of PACAP-38 on the oxidative stress and tissue damage in autotransplanted intestine. Sham-operated, ischemia/reperfusion, and autotransplanted groups were established in Wistar rats. In ischemia/reperfusion groups, 1 h (group A), 2 h (group B), and 3 h (group C) ischemia followed by 3 h of reperfusion was applied. In autotransplanted groups, total orthotopic intestinal autotransplantation was performed. Grafts were preserved in University of Wisconsin (UW) solution and in UW containing 30 microg PACAP-38 for 1, 2, 3, and 6 h. Reperfusion lasted 3 h in all groups. Endogenous PACAP-38 concentration was measured by radioimmunoassay. To determine oxidative stress parameters, malondialdehyde, reduced glutathione, and superoxide dismutase were measured in tissue samples. Tissue damage was analyzed by qualitative and quantitative methods on hematoxylin/eosin-stained sections. Concentration of endogenous PACAP-38 significantly decreased in groups B and C compared to sham-operated group. Preservation solution containing PACAP-38 ameliorated bowel tissue oxidative injury induced by cold ischemia and reperfusion. Histological results showed that preservation caused destruction of the mucous, submucous, and muscular layers, which were further deteriorated by the end of reperfusion. In contrast, PACAP-38 significantly protected the intestinal structure. Ischemia/reperfusion decreased the endogenous PACAP-38 concentration in the intestinal tissue. Administration of PACAP-38 mitigated the oxidative injury and histological lesions in small-bowel autotransplantation model.

Design, Synthesis, and Preclinical Efficacy of Novel Nonretinoid Antagonists of Retinol-Binding Protein 4 in the Mouse Model of Hepatic Steatosis.

Retinol-binding protein 4 (RBP4) serves as a transporter for all- trans-retinol (1) in the blood, and it has been proposed to act as an adipokine. Elevated plasma levels of the protein have been linked to diabetes, obesity, cardiovascular diseases, and nonalcoholic fatty liver disease (NAFLD). Recently, adipocyte-specific overexpression of RBP4 was reported to cause hepatic steatosis in mice. We previously identified an orally bioavailable RBP4 antagonist that significantly lowered RBP4 serum levels in Abca4-/- knockout mice with concomitant normalization of complement system protein expression and reduction of bisretinoid formation within the retinal pigment epithelium. We describe herein the discovery of novel RBP4 antagonists 48 and 59, which reduce serum RBP4 levels by >80% in mice upon acute oral dosing. Furthermore, 59 demonstrated efficacy in the transgenic adi-hRBP4 murine model of hepatic steatosis, suggesting that RBP4 antagonists may also have therapeutic utility for the treatment of NAFLD.

Effects of PACAP on survival and renal morphology in rats subjected to renal ischemia/reperfusion.

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs and exerts a variety of biological functions in the nervous system and in the peripheral organs, including the urinary system. PACAP has protective effects against myeloma kidney injury and renal ischemia. Ischemia/reperfusion injury of the kidney is a major clinical problem, and based on the protective effects of PACAP in cerebral and cardiomyocyte ischemia, the aim of the present study was to evaluate the effects of a single intravenous PACAP injection on the survival and renal morphology after varying times of ischemia. Rats were subjected to renal artery clamping for 15, 30, 45, 60, or 75 min followed by reperfusion. PACAP (100 microg) was administered intravenously before arterial clamping. We found that a 15- or 30-min renal ischemia led to no renal dysfunction, and the kidneys showed normal appearance with no difference between PACAP- and saline-treated groups. Control rats with 45 min of ischemia had increased premature death rate and showed multifocal acute tubular atrophy, while a 60-min ischemia led to death of all control animals within a few days displaying severe, multifocal Grade II tubular atrophy. In contrast, all PACAP-treated animals survived with subtle morphological changes after the 45-min ischemia. After the 60-min ischemia, death rate was significantly lower in PACAP-treated rats compared to controls, and animals showed subtle focal tubular alteration. A 75-min ischemia was not performable in controls because of deaths before the termination of ischemia. PACAP-treated rats survived longer, but they also died after 5-10 days exhibiting severe focal tubular atrophy. In summary, our results clearly show that PACAP is able to prolong the renal ischemic time, decrease mortality, and attenuate tubular degeneration after renal ischemia.

Effects of PACAP on the circadian changes of signaling pathways in chicken pinealocytes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of circadian rhythms. In mammals, the brain's biological clock is the suprachiasmatic nucleus, receiving photic information from the retina through the retinohypothalamic pathway, where PACAP is the main cotransmitter of glutamate. The primary conductor of circadian rhythms of birds is the pineal gland. The presence of PACAP has been demonstrated both in the rat and avian pineal gland, where PACAP stimulates melatonin synthesis. The signaling mechanism, by which PACAP modulates melatonin synthesis and circadian rhythmic functions of the pineal gland, is only partially known. The aim of the present study was to investigate the effects of PACAP on the changes of p38 mitogen-activated protein kinase (MAPK) and 14-3-3 protein in chick pineal cell culture both of which have been shown to participate in the regulation of rhythmic functions. Pineal cells were treated with 1, 10, or 100 nM PACAP38 every 4 h during a 24-h period. The phosphorylation of p38 MAPK showed obvious changes during the observed 24 h, while the level of 14-3-3 protein did not. We found that the lowest used dose of PACAP did not cause any phase alteration in p38 MAPK phosphorylation. Ten nM PACAP induced a 4-h-long delay and 100 nM abolished the circadian changes of p38 MAPK phosphorylation. PACAP was not effective on the level of 14-3-3 protein in the early morning hours, and only the highest tested dose (100 nM) could evoke a change in the appearance of 14-3-3 between midday and midnight hours. In summary, PACAP modulated the phosphorylation of p38 MAPK and the appearance of 14-3-3 protein in the chicken pineal cells, but these effects were dose dependent and also depended on the time of day.

Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes.

Articular chondrocytes have a well-developed osmoregulatory system that enables cells to survive in a constantly changing osmotic environment. However, osmotic loading exceeding that occurring under physiological conditions severely compromises chondrocyte function and leads to degenerative changes. The aim of the present study was to investigate the form of cell death and changes in apoptotic signaling pathways under hyperosmotic stress using a primary chondrocyte culture. Cell viability and apoptosis assays performed with annexin V and propidium iodide staining showed that a highly hyperosmotic medium (600 mOsm) severely reduced chondrocyte viability and led mainly to apoptotic cell death, while elevating osmotic pressure within the physiological range caused no changes compared to isosmotic conditions. Western blot analysis revealed that a 600 mOsm hyperosmotic environment induced the activation of proapoptotic members of the mitogen-activated protein kinase family such as c-Jun N-terminal kinase (JNK) and p38, and led to an increased level of extracellular signal regulated kinase (ERK1/2). Hyperosmotic stress also induced the activation of caspase-3. In summary, our results show that hyperosmotic stress leads to mainly apoptotic cell death via the involvement of proapoptotic signaling pathways in a primary chondrocyte culture.

Expression and protective role of heme oxygenase-1 in delayed myocardial preconditioning.

In the study the authors aimed to demonstrate the expression and protective effect of heme oxygenase-1 (HO-1) in the delayed preconditioning (PC) on cultured myocardiac cells. Neonatal rat cardiac myocytes were exposed to ischemic (ischemic medium [IM] for 20 min) and pharmacological (adenosine, epinephrine, opioid) PC. Twenty-four hours later cells were subjected to a simulated ischemia (SI)--culturing for 3 h in IM, followed by 2-h reperfusion in normal medium--and then lactate dehydrogenase (LDH), live/death ratio, and apoptosis were measured. For demonstrating the protective role of HO-1, its enzymatic activity was competitively inhibited by administration of zinc protoporphyrin IX (ZnPPIX), and HO-1 synthesis was blocked with HO-1 siRNA. Cells in control group were cultured under normoxic conditions. In SI group, cells underwent only an SI without PC. HO-1 expression in all of the groups was demonstrated with immunostaining. Our results showed a significant decrease of LDH release, apoptosis, and cell death in PC groups versus SI group, which has been risen in ZnPPIX- and HO-1 siRNA-treated groups. HO-1 immunostaining showed an appreciable HO-1 expression in PC groups, which was abolished with HO-1 siRNA administration, but not in ZnPPIX group. The results therefore suggest that HO-1 expression increases in both ischemic and pharmacological PC, and HO-1 has cellular protective effect against cell death and apoptosis in ischemia-reperfusion-induced oxidative injury.

Cardioprotective action of urocortin in early pre- and postconditioning.

Pre- and postconditioning are powerful endogenous adaptive phenomenon of the organism whereby different stimuli enhance the tolerance against various types of stress. Urocortin (Ucn), member of the corticotropin-releasing factor (CRF) family has potent effects on the cardiovascular system. The aim of this article was to investigate the action of Ucn on cultured cardiomyocytes in the process of pre- and postconditioning. Isolated neonatal rat ventricular myocytes were preconditioned with adenosine, simulated ischemia, and Ucn (10-min treatment followed by 10-min reperfusion/recovery). For detecting the effect of alternative types of preconditioning, necrosis enzyme (lactate dehydrogenase [LDH]) release, vital staining (trypan blue), and ratio of apoptosis/necrosis were examined after cardiac cells were exposed to 3-h sustained ischemia and 2-h reperfusion. Same parameters were measured in the postconditioned groups (30- or 60-min ischemia followed by postconditioning with 10-min ischemic stimulus or Ucn and 2-h reperfusion). Cells exposed to 3-h ischemia followed by 2-h reperfusion were shown as control. Our results show that LDH release a number of trypan blue-stained dead cells and the ratio of apoptotized and necrotized cells was decreased in all preconditioned groups compared with control group. In postconditioned groups LDH content of culture medium, trypan blue-positive cardiomyocytes, and the rate of apoptotic/necrotic cells was reduced contrasted with non-postconditioned group. We can conclude that preconditioning with Ucn induced such a powerful cell protective effect as adenosine and ischemia. Furthermore, postconditioning with Ucn after 60-min ischemia was more cardioprotective than ischemic postconditioning.

Effects of pituitary adenylate cyclase activating polypeptide (PACAP) on the PKA-Bad-14-3-3 signaling pathway in glutamate-induced retinal injury in neonatal rats.

The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) and its receptors are widely expressed in the nervous system including the retina. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently, we have shown that PACAP1-38 is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. Studying the molecular mechanisms of this protection has revealed that PACAP1-38 stimulates anti-apoptotic mechanisms such as phosphorylation of ERK1/2 and inhibits pro-apoptotic signaling molecules such as JNK1/2, p38MAPK, caspase-3 and the translocation of mitochondrial cytochrome c and apoptosis inducing factor in glutamate-treated retinas in vivo. In the present study we investigated the effects of PACAP1-38 on a further signal transduction pathway possibly involved in the protective effect of intravitreal PACAP1-38 administration against apoptotic retinal degeneration induced by neonatal MSG treatment. The focus of the present study was the protein kinase A (PKA)-Bad-14-3-3 transduction pathway. In vivo MSG treatment led to a reduction in the levels of anti-apoptotic molecules (phospho-PKA phospho-Bad, Bcl-xL and 14-3-3 proteins) in the retina. Co-treatment with PACAP1-38 counteracted these effects: the level of phospho-PKA, phospho-Bad, Bcl-xL and 14-3-3 were increased. All effects of PACAP1-38 were inhibited by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP1-38 activates the PKA-Bad-14-3-3 pathway which is inhibited by MSG treatment. Our results also provide new insights into the signaling mechanisms possibly involved in the PACAP-mediated anti-apoptotic effects.

PARP inhibition protects mitochondria and reduces ROS production via PARP-1-ATF4-MKP-1-MAPK retrograde pathway.

Oxidative stress induces DNA breaks and PARP-1 activation which initiates mitochondrial reactive oxygen species (ROS) production and cell death through pathways not yet identified. Here, we show the mechanism by which PARP-1 influences these processes via PARylation of activating transcription factor-4 (ATF4) responsible for MAP kinase phosphatase-1 (MKP-1) expression and thereby regulates MAP kinases. PARP inhibitor, or silencing, of PARP induced MKP-1 expression by ATF4-dependent way, and inactivated JNK and p38 MAP kinases. Additionally, it induced ATF4 expression and binding to cAMP-response element (CRE) leading to MKP-1 expression and the inactivation of MAP kinases. In contrast, PARP-1 activation induced the PARylation of ATF4 and reduced its binding to CRE sequence in vitro. CHIP-qPCR analysis showed that PARP inhibitor increased the ATF4 occupancy at the initiation site of MKP-1. In oxidative stress, PARP inhibition reduced ROS-induced cell death, suppressed mitochondrial ROS production and protected mitochondrial membrane potential on an ATF4 and MKP-1 dependent way. Basically identical results were obtained in WRL-68, A-549 and T24/83 human cell lines indicating that the aforementioned mechanism can be universal. Here, we provide the first description of PARP-1-ATF4-MKP-1-JNK/p38 MAPK retrograde pathway, which is responsible for the regulation of mitochondrial integrity, ROS production and cell death in oxidative stress, and may represent a new mechanism of PARP in cancer therapy since cancer stem cells development is JNK-dependent.

PACAP inhibits oxidative stress-induced activation of MAP kinase-dependent apoptotic pathway in cultured cardiomyocytes.

The present article investigated the effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on oxidative stress-induced apoptosis in neonatal rat cardiomyocytes. Our results show that PACAP decreased the ratio of apoptotic cells following H2O2 treatment. PACAP also diminished the activity of apoptosis signal-regulating kinase. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis and suggest that its cardioprotective effect is mediated through inhibition of the MAP kinase-dependent apoptotic pathway.