Rolipram and Pentoxifylline Combination Ameliorates Experimental Diabetic Neuropathy Through Inhibition of Oxidative Stress and Inflammatory Pathways in the Dorsal Root Ganglion Neurons

Diabetic neuropathy (DN) is the most challenging microvascular complication of diabetes and there is no suitable treatment for it, so the development of new agents to relieve DN is urgently needed. Since oxidative stress and inflammation play an essential role in the development of DN, clearance of these factors are good strategies for the treatment of this disease. According to key role of cyclic adenosine monophosphate (cAMP) in the regulation of oxidative stress and inflammatory pathways, it seems that phosphodiesterase inhibitors (PDEIs) can be as novel drug targets for improving DN through enhancement of cAMP level. The aim of this study was to evaluate the effects of rolipram, a selective PDE4 inhibitor, and pentoxifylline, a general PDE inhibitor on experimental model of DN and also to determine the possible mechanisms involved in the effectiveness of these agents. We investigated the effects of rolipram (1mg/kg) and pentoxifylline (100 mg/kg) and also combination of rolipram (0.5 mg/kg) and pentoxifylline (50 mg/kg), orally for five weeks in rats that became diabetic by STZ (55 mg/kg, i.p.). After treatments, motor function was evaluated by open-field test, then rats were anesthetized and dorsal root ganglion (DRG) neurons isolated. Next, oxidative stress biomarkers and inflammatory factors were assessed by biochemical and ELISA methods, and RT-PCR analysis in DRG neurons. Rolipram and/or pentoxifylline treatment significantly attenuated DN – induced motor function deficiency by modulating distance moved and velocity. Rolipram and/or pentoxifylline treatment dramatically increased the cAMP level, as well as suppressed DN – induced oxidative stress which was associated with decrease in LPO and ROS and increase in TAC, total thiol, CAT and SOD in DRG neurons. On the other hand, the level of inflammatory factors (TNF-α, NF-kB and COX2) significantly decreased following rolipram and/or pentoxifylline administration.The maximum effectiveness was with rolipram and/or pentoxifylline combination on mentioned factors.These findings provide novel experimental evidence for further clinical investigations on rolipram and pentoxifylline combination for the treatment of DN.

Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins

Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER–mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.

A Bumpy Ride of Mycobacterial Phagosome Maturation: Roleplay of Coronin1 Through Cofilin1 and cAMP

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.

Differential receptor crosstalk in DRD1-DRD2 heterodimer upon phasic and tonic dopamine signals

In response to phasic and tonic release, dopamine neurotransmission is regulated by its receptor subtypes, mainly dopamine receptor type 1 and 2 (DRD1 and DRD2). These dopamine receptors are known to form a heterodimer, however the receptor crosstalk between DRD1 and DRD2 was only suspected by measuring their downstream signaling products, due to the lack of methodology for selectively detecting individual activity of different dopamine receptors. Here, we develop red DRD1 sensor (R-DRD1) and green DRD2 sensor (G-DRD2) which can specifically monitor the real-time activity of DRD1 and DRD2, and apply these multicolor sensors to directly measure the receptor crosstalk in the DRD1-DRD2 heterodimer. Surprisingly, we discover that DRD1 activation in the heterodimer is inhibited only at micromolar phasic concentration of dopamine, while DRD2 activation is selectively inhibited at nanomolar tonic dopamine level. Differential receptor crosstalk in the DRD1-DRD2 heterodimer further modulates their downstream cAMP level. These data imply a novel function of the DRD1-DRD2 heterodimer at physiological dopamine levels of phasic and tonic release. Our approach utilizing multicolor receptor sensors will be useful to discover novel function of GPCR heterodimers.

In-vitro Study: Camp Overshoot Caused by Chronic Morphine Alleviated by the Synergistic Combination of Zamzam Water and Methadone in Human Primary Glioblastoma Cell Line (U-87 MG)

Introduction: Zamzam water is blessed water originated from Mecca and is believed by Muslims to have the ability to cure illness. This ability to cure illness is due to the fact that zamzam water has higher concentration of minerals especially sodium, calcium and magnesium which play a vital role. Sodium is reported to be involved in the regulation of the Mu-Opioid Receptor, which indirectly leads to the production of cAMP. Hence, this current study was carried out to investigate the synergistic effects of zamzam water and methadone combination on cAMP levels in human primary glioblastoma cell line (U-87 MG) after chronic morphine administration. Materials and Methods: The time course and concentration of morphine on U-87 MG cell line was determined. The U-87 MG cell line was incubated with morphine (25 µL/mL) for 24h, to make the cell dependent on morphine and later treated with different combinations of 3.2 mL of zamzam water and methadone (5, 10, 25 µL/mL). The levels of cAMP was determined using the enzyme-linked immunosorbent assay kit. Results: The result revealed that 3.2 mL of zamzam water incubated with 10 µl/ mL of methadone significantly prevented the overshoot production of cAMP level (p<0.05) in U-87 MG cell line after 48h incubation when compared to the untreated samples. Conclusion: These finding suggest that co-treatment with zamzam water and methadone could possibly avoid tolerance and dependence on chronic morphine treatment by preventing the up-regulation of cAMP level.><0.05 in U-87 MG cell line after 48h incubation when compared to the untreated samples. Conclusion: These finding suggest that co-treatment with zamzam water and methadone could possibly avoid tolerance and dependence on chronic morphine treatment by preventing the up-regulation of cAMP level.

Role of phosphodiesterases in the development of takotsubo syndrome

Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. We identified a higher sensitivity to catecholamine-induced stress toxicity as mechanism associated with the TTS phenotype in our former study, but the pathogenesis of TTS is still not completely understood. In this study our aim was to prove the hypothesis of an altered phosphodiesterase (PDE)-dependent 3',5'-cyclic adenosine monophosphate (cAMP)-signaling in TTS in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods and results We generated functional TTS-iPSC-CMs and treated them with catecholamines to mimic a TTS-phenotype. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used Förster resonance energy transfer (FRET) based cAMP sensors, which are specifically located in the cytosol or at the sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA) micro domain. We demonstrated that β-adrenergic receptor (β-AR) stimulations resulted in stronger cytosolic FRET responses in TTS-CMs compared to controls. In contrast, no differences of cAMP level were observed in the SERCA-PLN micro domain between TTS- and control-iPSC-CMs. To analyze the interplay of β-AR signaling and specific PDE contribution to the cAMP signaling in TTS, specific PDE-inhibitors were used. We were able to show in the cytosol that after β-AR stimulation, the strong effects of the PDE4 family of control cells were significantly decreased in diseased TTS CMs, which is in line with previously described reduced PDE4 activity in failing mouse hearts. In contrast, the contribution of PDE3 to cytoplasmic cAMP degradation was increased in TTS (Figure 1 A). This is in line with increased PDE3A and down-regulated PDE4D protein expression in TTS-iPSC-CMs compared to control cells. Analysis of PDE-dependent cAMP level in the SERCA micro domain show also a significantly reduced PDE4 activity. But the dynamic cytosolic PDE contribution of PDE2 and PDE3 after catecholamine treatment in TTS is lost in SERCA micro domain (Figure1B). Conclusion Our data showed for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. We demonstrated an isozym shift from PDE4 in control to PDE3 and PDE2 in TTS and identified PDE4 as an important player in the β-adrenergic cAMP signaling in TTS. Therefore, PDE4 activators may be a possible new therapeutic target option in the treatment of TTS. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DZHK

Protective Effects of Adiponectin against Cobalt Chloride-Induced Apoptosis of Smooth Muscle Cells via cAMP/PKA Pathway

Adiponectin (APN) is an adipokine secreted from adipose tissue and exhibits biological functions such as microcirculation-regulating, hearing-protective, and antiapoptotic. However, the effect of APN on the apoptosis of spiral arterial smooth muscle cells (SMCs) under hypoxic conditions in vitro is not clear. We used cobalt chloride (CoCl2) to simulate chemical hypoxia in vitro, and the SMCs were pretreated with APN and then stimulated with CoCl2. The viability of cells and apoptosis were assessed by CCK-8 and flow cytometry, respectively. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, cAMP level, and the activity of PKA were detected by ELISA. Protein expression and localization were studied by Western blot and immunofluorescence analysis. In the present study, we found that APN exhibits antiapoptosis effects. CoCl2 exhibited decreased cell viability, increased apoptosis and MDA levels, and decreased SOD activity in a concentration-dependent manner, compared with the control group. Moreover, CoCl2 upregulated the expression levels of Bax and cleaved caspase-3 and then downregulated Bcl-2 levels in a time-dependent manner. Compared with the CoCl2 group, the group pretreated with APN had increased cell viability, SOD activity, PKA activity, cAMP level, and PKA expression, but decreased MDA levels and apoptosis. Lastly, the protective effect of APN was blocked by cAMP inhibitor SQ22536 and PKA inhibitor H 89. These results showed that APN protected SMCs against CoCl2-induced hypoxic injury via the cAMP/PKA signaling pathway.

PDE2A Is Indispensable for Mouse Liver Development and Hematopoiesis

Phosphodiesterase 2A (PDE2A) is a cAMP-cGMP hydrolyzing enzyme essential for mouse development and the PDE2A knockout model (PDE2A−/−) is embryonic lethal. Notably, livers of PDE2A−/− embryos at embryonic day 14.5 (E14.5) have extremely reduced size. Morphological, cellular and molecular analyses revealed loss of integrity in the PDE2A−/− liver niche that compromises the hematopoietic function and maturation. Hematopoietic cells isolated from PDE2A−/− livers are instead able to differentiate in in vitro assays, suggesting the absence of blood cell-autonomous defects. Apoptosis was revealed in hepatoblasts and at the endothelial and stromal compartments in livers of PDE2A−/− embryos. The increase of the intracellular cAMP level and of the inducible cAMP early repressor (ICER) in liver of PDE2A−/− embryos might explain the impairment of liver development by downregulating the expression of the anti-apoptotic gene Bcl2. In summary, we propose PDE2A as an essential gene for integrity maintenance of liver niche and the accomplishment of hematopoiesis.