cAMP Content Research Articles

Inhibitory effects of a maleimide compound on the virulence factors of Candida albicans

ABSTRACT Candidiasis caused by Candida albicans infection has long been a serious human health problem. The pathogenicity of C. albicans is mainly due to its virulence factors, which are novel targets of antifungal drugs for low risk of resistance development. In this study, we identified a maleimide compound [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] that exerts effective anti-virulence activity. It could inhibit the process of adhesion, filamentation, and biofilm formation in C. albicans. In addition, it exhibited low cytotoxicity, hemolytic activity, and drug resistance development. Moreover, in Galleria mellonella-C. albicans (in vivo) infection model, the survival time of infected larvae was significantly prolonged under the treatment of MPD. Further, mechanism research revealed that MPD increased farnesol secretion by upregulating the expression of Dpp3. The increased farnesol inhibited the activity of Cdc35, which then decreased the intracellular cAMP content resulting in the inhibition of virulence factors via the Ras1-cAMP-Efg1 pathway. In all, this study evaluated the inhibitory effect of MPD on various virulence factors of C. albicans and identified the underlying mechanisms. This suggests a potential application of MPD to overcome fungal infections in clinics.

Multiple-Factors-Induced Rheumatoid Arthritis Synoviocyte Activation Is Attenuated by the α2-Adrenergic Receptor Agonist Dexmedetomidine.

Dexmedetomidine (Dex) has analgesic and sedative properties and anti-inflammatory functions. Although the effects of Dex on arthritis have been revealed, the physiological mechanism underlying the interaction between Dex and rheumatoid arthritis (RA)-mediated inflammatory cytokines has not been fully studied. Inflamed and migrated fibroblast-like synoviocytes (FLSs) are involved in RA severity. Thus, we aimed to determine the effects of Dex on RA-FLSs treated with inflammatory cytokines and a growth factor as multiple stimulating inputs. TNF-α, IL-6, and EGF as multiple stimulating inputs increased the cAMP concentration of RA-FLSs, while Dex treatment reduced cAMP concentration. Dex reduced electroneutral sodium-bicarbonate cotransporter 1 (NBCn1) expression, NBC activity, and subsequent RA-FLS migration. The mRNA expression levels of RA-related factors, such as inflammatory cytokines and osteoclastogenesis factors, were enhanced by multiple-input treatment. Notably, Dex effectively reduced these expression levels in RA-FLSs. These results indicate that multiple inflammatory or stimulating inputs enhance RA-FLS migration, and treatment with Dex relieves activated RA-FLSs, suggesting that Dex is a potential therapeutic drug for RA.

Luman regulates the activity of the LHCGR promoter

Testosterone in male mammals is mainly secreted by testicular Leydig cells, and its secretion process is regulated by the hypothalamic-pituitary-gonadal axis. After receiving the luteinizing hormone (LH) stimulus signal, the lutropin/choriogonadotropin receptor (LHCGR) on the Leydig cell membrane transfers the signal into the cell and finally increases the secretion of testosterone by upregulating the expression of steroid hormone synthase. In previous experiments, we found that interfering with the expression of the Luman protein can significantly increase testosterone secretion in MLTC-1 cells. In this experiment, we found that knockdown of Luman in MLTC-1 cells significantly increased the concentration of cAMP and upregulated the expression of AC and LHCGR. Moreover, an analysis of the activity of the LHCGR promoter by a dual luciferase reporter system showed that knockdown of Luman increased the activity of the LHCGR promoter. Therefore, we believe that knockdown of Luman increased the activity of the LHCGR promoter and upregulated the expression of LHCGR, thereby increasing the concentration of intracellular cAMP and ultimately leading to an increase of testosterone secretion by MLTC-1 cells.

Potential for modulation of platelet function via adenosine receptors during inflammation.

Traditionally, platelets are known to play an important role in haemostasis and thrombosis; however, they serve also as important modulators of inflammation and immunity. Platelets secrete adhesion molecules and cytokines, interact with leukocytes and endothelium, and express toll-like receptors involved in a direct interaction with pathogens. Platelets express A2A and A2B subtypes of receptors for adenosine. The activation of these receptors leads to an increase in cAMP concentration in the cytoplasm, thereby resulting in inhibited secretion of pro-inflammatory mediators and reduced cell activation. Therefore, platelet adenosine receptors could be a potential target for inhibiting platelet activation and thus down-regulating inflammation or immunity. The biological effects of adenosine are short-lasting, because the compound is rapidly metabolized; hence, its lability has triggered efforts to synthesize new, longer-lasting adenosine analogues. In this article, we have reviewed the literature regarding the pharmacological potential of adenosine and other agonists of A2A and A2B receptors to affect platelet function during inflammation. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Protein interactome of 3',5'-cyclic adenosine monophosphate reveals its role in regulating the actin cytoskeleton.

Identification of protein interactors is ideally suited for the functional characterization of small molecules. Cyclic adenosine monophosphate (3',5'-cAMP) is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3',5'-cAMP, we used a novel chemo-proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3',5'-cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3',5'-cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3',5'-cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3',5'-cAMP supplementation affected actin organization by inducing actin-bundling. Consistent with these results, the increase in 3',5'-cAMP levels, obtained either by feeding or by chemical modulation of 3',5'-cAMP metabolism was sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. The observed rescue was specific to 3',5'-cAMP, as demonstrated using a positional isomer 2',3'-cAMP, and true for the nanomolar 3',5'-cAMP concentrations reported for plant cells. In vitro characterization of the 3',5'-cAMP - actin pairing argues against a direct interaction between actin and 3',5'-cAMP. Alternative mechanisms by which 3',5'-cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a novel resource, 3',5'-cAMP interactome, and a new functional insight into the 3',5'-cAMP-mediated regulation in plants.

Phosducin-like protein PoPlp1 impacts cellulase and amylase expression and development in Penicillium oxalicum via the G protein-cAMP signaling pathway.

In this study, a phosducin-like protein, PoPlp1, was identified and functionally studied in the cellulase-producing strain Penicillium oxalicum 114-2. PoPlp1 was proven to participate in several biological processes, including mycelium development, conidiation, and expression of cellulases and amylases. With deletion of Poplp1, morphology and development varied significantly in ΔPoplp1. Colony growth, glucose utilization, and the hydrolysis capability of starch and cellulose were limited, whereas conidiation was enhanced. Based on detection of the levels of expression of transcription factors involved in asexual development, we conjectured that PoPlp1 is involved in conidiation via the major factor BrlA. We explored the effect of PoPlp1 on cellulase and amylase expression and observed that cellulase and amylase activity and major gene transcription levels were all dramatically reduced in ΔPoplp1. Deletion of PoPlp1 caused a decrease in intracellular cAMP levels, and the cellulase gene expression level of ΔPoplp1 was restored to a certain extent through external addition of cAMP. These findings demonstrate that PoPlp1 may affect cellulase and amylase expression by regulating cAMP concentration. To comprehensively explore the mechanism of PoPlp1 in regulating multiple biological processes, we performed a comparative transcriptomic analysis between strains P. oxalicum 114-2 and ΔPoplp1. The major cellulase and amylase genes were all downregulated, congrent with the results of real-time quantitative polymerase chain reaction analysis. The genes involved in the G protein-cAMP signaling pathway, including several G-protein-coupled receptors, one regulator of G protein signaling, and two cAMP phosphodiesterases, were disrupted by deletion of PoPlp1. These results confirm the positive function of PoPlp1 in the G protein-cAMP signaling pathway. This functional analysis of PoPlp1 will be very beneficial for further study of the regulatory mechanisms of cellulase expression and other biological processes in P. oxalicum 114-2 via the G protein-cAMP signaling pathway.

Underlying mechnism of electroacupuncture for treating detrusor hyperreflexia after suprasacral spinal cord injury through the PACAP-cAMP signaling pathway: 基于PACAP-cAMP信号通路研究电针治疗骶上脊髓损伤后逼尿肌反射亢进型膀胱的效应机制

ObjectiveTo elucidate the underlying mechanism and effect of electroacupuncture (EA) on the neurogenic bladder following suprasacral spinal cord injury (SSCI). A rat model of detrusor hyperreflexia after SSCI was established to examine the urodynamics, detrusor muscle tissue morphology, the protein and mRNA expression levels of pituitary adenylate cyclase activating peptide (PACAP) and its receptor PAC1R, and cyclic adenosine monophosphate (cAMP) content in the detrusor muscle with a focus on the PACAP-cAMP signaling pathway. MethodA total of 72 female SD rats were randomized into control group and sham operation group (n=12 per group) by using a random number table. The remaining 48 rats were established into the model of detrusor hyperreflexia after SSCI. After successful modeling, these rats were randomly assigned to model, EA, and EA + PACAP6-38 groups (n=12 per group). The unsuccessful modeled rats were used for exploratory observation. For the rats in EA group, “Ciliao (BL32)” “Zhongji (CV3)”, and “Sanyinjiao (SP6)” were needled and stimulated by EA. The PACAP receptor antagonist PACAP6-38 was administered intraperitoneally in the EA + PACAP6-38 group before EA, and EA was applied for seven consecutive days. After treatment, the urodynamics of the rats were analyzed, and hematoxylin and eosin staining was used to examine rat bladder detrusor tissue morphology. The expressions of PACAP-38 and PAC1R were detected by immunohistochemistry and Western blot. The mRNA expression levels of PACAP-38 and PAC1R were examined by RT-qPCR, while cAMP content was detected by ELISA. Results(1) Compared with sham operation group, it was exhibited disarray in the transitional epithelium cells of the bladder in the modeled rats. The intercellular space was significantly widened, accompanied by inflammatory cell infiltration and noticeable tissue edema. Both the bladder initial pressure and leak point pressure of the rats were higher (P < 0.01), whereas the maximum cystometric capacity and bladder compliance were lower (P < 0.01). The protein and mRNA expression levels of PACAP-38 and PAC1R in the detrusor muscle, together with the cAMP content, were lower (P < 0.05). (2) Compared with the model rats, the EA group showed reduced inflammatory response in the detrusor muscle tissue, with decreased monocyte infiltration and less severe tissue edema. The bladder smooth muscle cells exhibited increased integrity, and there was decreased cellular tissue edema, inflammatory cell infiltration, and fibroplasia. The bladder initial pressure and leak point pressure were lower (P < 0.05), while the maximum cystometric capacity and bladder compliance were higher (P < 0.01). The protein and mRNA expression levels of PACAP-38 and PAC1R in the detrusor muscle, along with the cAMP content, were higher (P < 0.05). (3) Compared to the EA group, the EA + PACAP6-38 group showed a less organized arrangement of muscle fibers in the detrusor muscle tissue, larger intercellular space, monocyte infiltration, and considerable tissue edema. The changes in bladder initial pressure and leak point pressure were not significant (P > 0.05), while the maximum cystometric capacity and bladder compliance were lower (P < 0.05). The changes in the protein and mRNA expressions of PACAP-38 within the detrusor muscle were not significant (P > 0.05), whereas the protein and mRNA expressions of PAC1R were reduced (P < 0.05), and the cAMP content within the detrusor muscle was lower (P < 0.05). ConclusionEA can ameliorate the uninhibited contractile condition of the detrusor muscle in the bladder following SSCI. By mediating the PACAP-cAMP signaling pathway, it reduces the pathological damage to the detrusor muscle, thereby improving bladder function.

Role of the adenylate cyclase/cyclic AMP pathway in oxytocin-induced lacrimal gland myoepithelial cells contraction

The aim of these studies was to investigate the involvement of the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.

Metabolome shifts triggered by chlorine sanitisation induce Escherichia coli on fresh produce into the viable but nonculturable state

Facing the increasing occurrence of “big six” Escherichia coli outbreaks linked to fresh produce, chlorine-based sanitisers are widely used for fresh produce decontamination in recent years. However, latest finding that chlorine may induce E. coli cells into a viable not nonculturable (VBNC) state is bringing a new challenge to the fresh produce industry. VBNC cells are undetectable by the plate count test, and yet they retain pathogenicity and are more antibiotic-resistant than culturable cells. As a result, their eradication is critical to ensure the safety of fresh produce. Understanding VBNC cells at the metabolic level may provide a breakthrough for their eradication. Therefore, this study was carried out to collect the VBNC pathogenic E. coli (O26:H11, O121:H19, and O157:H7) cells from chlorine-treated pea sprouts and characterise them using NMR-based metabolomics. From the globally increased metabolite contents detected in the VBNC E. coli cells as compared to the culturable cells, mechanisms underlying E. coli’s VBNC induction were elucidated. These include rendering the energy generation scheme to become more compatible with the lowered energy needs, disaggregating protein aggregates to release amino acids for osmoprotection and later resuscitation, as well as increasing cAMP content to downregulate RpoS. These identified metabolic characteristics can inspire future development of targeted measures for VBNC E. coli cell inhibition. Our methods can also be applied to other pathogens to help lower the risk of overall foodborne diseases.

Mailuoning oral liquid attenuates convalescent cerebral ischemia by inhibiting AMPK/mTOR-associated apoptosis and promoting CREB/BDNF-mediated neuroprotection

Ethnopharmacological relevanceIschemic stroke is divided into acute, subacute and convalescent phases according to the time of onset. Clinically, Mailuoning oral liquid (MLN O) is a traditional Chinese patent medicine for treating ischemic stroke. Previous studies have shown that MLN O could prevent acute cerebral ischemia-reperfusion. However, its underlying mechanism remains unclear. Aim of the studyTo investigate the relationship between neuroprotection and apoptosis for clarifying MLN O mechanism in the recovery phase of ischemic stroke. Materials and methodsWe imitated stroke using middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro models. The infarct volume, neurological deficit scores, HE staining, Nissl staining, TUNEL staining, immunohistochemistry, and Western blot were correspondingly performed to find pathological changes and detect neuronal apoptosis in rat cerebral cortex. The contents of LDH, Cyt-c, c-AMP and BDNF in rat plasma and cerebral cortex were detected by ELISA. Cell viability was measured by CCK8 assay. Cell morphology, Hoechst 33342 staining and Annexin-V-Alexa Fluor 647/PI staining were performed to assess neuronal apoptosis. The expression levels of proteins were evaluated by western blotting. ResultsMLN O obviously reduced brain infarct volume and neurological deficit scores in MCAO rats. MLN O inhibited inflammatory cell infiltration and neuronal apoptosis, but promoted gliosis, neuronal survival, and neuroprotection in the cortical region of MCAO rats. Additionally, MLN O decreased the amount of LDH and cytochrome c, while increasing the expression of c-AMP in the plasma and ischemic cerebral cortex of MCAO rats, and promoting the expression of BDNF in the cortical tissue of MCAO rats. Besides, MLN O improved cell viability, restored cell morphology, while attenuating cell damage, inhibiting neuronal apoptosis following OGD/R in PC-12 cells. Moreover, MLN O inhibited apoptosis by suppressing the expression of pro-apoptotic-associated proteins, including Bax, cytochrome c, Cleaved caspase 3 and HIF-1α, whereas accelerating the expression of Bcl-2 in vivo and in vitro. Furthermore, MLN O inhibited the activity of AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR), but activated the signaling pathway of cAMP-response element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) in MCAO rats and OGD/R-stimulated PC-12 cells. ConclusionsThese results demonstrated that MLN O inhibited AMPK/mTOR to affect apoptosis associated with mitochondria, leading to improve CREB/BDNF-mediated neuroprotection in the recovery period of ischemic stroke in vivo and in vitro.

Study on the antipyretic mechanism of large pushing Tianheshui for young rabbits with endotoxin-induced fever

ObjectiveTo observe the effect of large pushing Tianheshui manipulation intervention on the body temperature of young rabbits with endotoxin-induced fever and discuss its antipyretic mechanism.MethodsThirty-two young rabbits meeting the standards were selected from 40 ordinary young male New Zealand rabbits after being adapted for 7 d, and randomly divided into a normal group, a model group, a large pushing Tianheshui group, and an ibuprofen group according to the random number table method, with 8 rabbits in each group. Rabbits in the model group, the large pushing Tianheshui group, and the ibuprofen group were subjected to preparing the endotoxin-induced fever model by intravenous lipopolysaccharide from the marginal ear vein. Rabbits in the large pushing Tianheshui group received Tuina (Chinese therapeutic massage) manipulation intervention 1.5 h and 2.5 h after modeling, respectively. Rabbits in the ibuprofen group were intragastrically given ibuprofen suspension 1.5 h after modeling. The dynamic changes in body temperature were observed for the young rabbits after fever modeling. Enzyme-linked immunosorbent assay was used to determine the content changes in positive mediators of hypothalamic body temperature, including prostaglandin (PG) E2 and cyclic adenosine monophosphate (cAMP), as well as negative mediators of hypothalamic body temperature, including arginine vasopressin (AVP) and α-melanocyte stimulating hormone (α-MSH).ResultsThe body temperature of the young rabbits in the model group was significantly higher than that in the normal group at 0.5–4.0 h, 5.0 h, and 5.5 h after modeling (P<0.01), showing two obvious fever peaks in the model group at 1.5 h and 3.0 h after modeling, respectively, with the highest peak at 1.5 h. Compared with the model group, body temperatures of the large pushing Tianheshui group and the ibuprofen group decreased significantly after 0.5 h of intervention (P<0.05). Compared with the normal group, the contents of PGE2 and cAMP were significantly increased (P<0.05), and the contents of AVP and α-MSH were significantly decreased (P<0.01) in the hypothalamus of the model group. Compared with the model group, the contents of PGE2 and cAMP were significantly decreased (P<0.01), and the levels of AVP and a-MSH were significantly increased (P<0.01) in the hypothalamus in the large pushing Tianheshui group and the ibuprofen group. There were no significant differences in the PGE2, cAMP, AVP, and α-MSH contents in the hypothalamus between the ibuprofen group and the large pushing Tianheshui group (P>0.05).ConclusionLarge pushing Tianheshui manipulation has a significant antipyretic effect on endotoxin-induced fever in young rabbits. The mechanism may be related to inhibiting the positive regulators (PGE2 and cAMP) and promoting the negative regulators (AVP and α-MSH) of hypothalamic control of body temperature.

3-Hydroxybutyrate ameliorates insulin resistance by inhibiting PPARγ Ser273 phosphorylation in type 2 diabetic mice

3-Hydroxybutyrate (3HB) is a small ketone body molecule produced endogenously by the body in the liver. Previous studies have shown that 3HB can reduce blood glucose level in type 2 diabetic (T2D) patients. However, there is no systematic study and clear mechanism to evaluate and explain the hypoglycemic effect of 3HB. Here we demonstrate that 3HB reduces fasting blood glucose level, improves glucose tolerance, and ameliorates insulin resistance in type 2 diabetic mice through hydroxycarboxylic acid receptor 2 (HCAR2). Mechanistically, 3HB increases intracellular calcium ion (Ca2+) levels by activating HCAR2, thereby stimulating adenylate cyclase (AC) to increase cyclic adenosine monophosphate (cAMP) concentration, and then activating protein kinase A (PKA). Activated PKA inhibits Raf1 proto-oncogene serine/threonine-protein kinase (Raf1) activity, resulting in a decrease in extracellular signal-regulated kinases 1/2 (ERK1/2) activity and ultimately inhibiting peroxisome proliferator-activated receptor γ (PPARγ) Ser273 phosphorylation in adipocytes. Inhibition of PPARγ Ser273 phosphorylation by 3HB altered the expression of PPARγ regulated genes and reduced insulin resistance. Collectively, 3HB ameliorates insulin resistance in type 2 diabetic mice through a pathway of HCAR2/Ca2+/cAMP/PKA/Raf1/ERK1/2/PPARγ.

Silver Carp (Hypophthalmichthys molitrix) Scale Collagen Peptides-1 (SCPs1) Inhibit Melanogenesis through Downregulation of the cAMP-CREB Signaling Pathway.

The mechanism of silver carp scale collagen peptides (SCPs1) on melanogenesis and its mechanism of action were examined in mouse melanoma cells (B16). The cell viability and effects of SCPs1 on intracellular tyrosinase (TYR) activity and melanin, reactive oxygen species (ROS), glutathione (GSH) and cyclic adenosine monophosphate (cAMP) content were examined. The regulatory mechanism of SCPs1 on the cAMP response element-binding protein (CREB) signaling pathway was analyzed. The cell viability of the SCPs1 group was >80% (0.01-1 mg/mL) and the inhibitory rate of SCPs1 on B16 cell melanin increased in a dose-dependent manner. The highest inhibitory rate of SCPs1 on melanin content reaching 80.24%. SCPs1 significantly increased the GSH content and decreased the tyrosinase activity, as well as the content of ROS and cAMP. Western blot analysis showed that SCPs1 significantly inhibited melanocortin-1 receptor (MC1R) expression and CREB phosphorylation in the cAMP-CREB signaling pathway, leading to downregulation of microphthalmia-associated transcription factor (MITF) and the expression of TYR, TYR-related protein-1 (TRP-1) and TRP-2. SCPs1 also inhibited the expression of MC1R, MITF, TYR, TRP-1 and TRP-2 at the transcriptional level. Taken together, SCPs1 inhibited melanin synthesis through the downregulation of the cAMP-CREB signaling pathway. Fish-derived collagen peptides could potentially be applied in skin whitening products.

Network pharmacology and molecular docking prediction, combined with experimental validation to explore the potential mechanism of Qishen Yiqi pills against HF-related cognitive dysfunction

Ethnopharmacological relevanceQishen Yiqi Pills (QSYQ) is a classical herbal formula for treating heart failure (HF) and has potential efficacy in improving cognitive function. The latter is one of the most common complications in patients with HF. However, there is no study on treating HF-related cognitive dysfunction by QSYQ. Aims of the studyThe study aims to investigate the effect and mechanism of QSYQ on treating post-HF cognitive dysfunction based on network pharmacology and experimental validation. Materials and methodsNetwork pharmacology analysis and molecular docking was used to explore endogenous targets of QSYQ in treating cognitive impairment. Ligation of the anterior descending branch of the left coronary artery and sleep deprivation (SD) were used to induce HF-related cognitive dysfunction in rats. The efficacy and potential signal targets of QSYQ were then verified by functional evaluation, pathological staining, and molecular biology experiments. Results384 common targets were identified by intersecting QSYQ ‘compound targets’ and ‘cognitive dysfunction’ disease targets. KEGG analysis showed these targets were enriched to the cAMP signal, and four marks responsible for regulating the cAMP signal were successfully docked with core compounds of QSYQ. Animal experiments demonstrated that QSYQ significantly ameliorated cardiac function and cognitive function in rats suffering from HF and SD, inhibited the reduction of cAMP and BDNF content, reversed the upregulation of PDE4 and downregulation of CREB, suppressed the loss of neurons, and restored the expression of synaptic protein PSD95 in the hippocampus. ConclusionThis study clarified that QSYQ could improve HF-related cognitive dysfunction by modulating cAMP-CREB-BDNF signals. It provides a rich basis for the potential mechanism of QSYQ in the treatment of heart failure with cognitive dysfunction.

SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+-dependent manner by cAMP and ATP in intestinal epithelial cells.

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl-/[Formula: see text] exchanger that is part of neutral NaCl absorption under baseline conditions, but in cyclic adenosine monophosphate (cAMP)-driven diarrheas, it is stimulated and contributes to increased anion secretion. To further understand the regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and adenosine 5'-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 µM and ATP at 0.25 µM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP increased intracellular Ca2+ (Ca2+i) in a concentration-dependent manner, whereas FSK (1 µM), which by itself did not significantly alter Ca2+i, followed by 0.25 µM ATP produced a large increase in Ca2+ that was approximately equal to the elevation caused by 1 µM ATP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) pretreatment prevented the ATP and FSK/ATP synergistically increased the DRA activity and the increase in Ca2+i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca2+) synergistically increased Ca2+i and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases, such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, whereas separation of DRA from Na+/H+ exchanger isoform-3 (NHE3) contributes to reduced NaCl absorption.NEW & NOTEWORTHY The BB Cl-/[Formula: see text] exchanger DRA takes part in both neutral NaCl absorption and stimulated anion secretion. Using intestinal cell line, Caco-2/BBE high concentrations of cAMP and Ca2+ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca2+. This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca2+ are involved.

A Novel Flow Cytometry-Based Assay for the Identification of HCN4 CNBD Ligands.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are promising therapeutic targets because of their association with the genesis of several diseases. The identification of selective compounds that alter cAMP-induced ion channel modulation by binding to the cyclic nucleotide-binding domain (CNBD) will facilitate HCN channel-specific drug development. In this study, a fast and protein purification-free ligand-binding approach with a surface-displayed HCN4 C-Linker-CNBD on E. coli is presented. 8-Fluo-cAMP ligand binding was monitored by single-cell analysis via flow cytometry, and a Kd-value of 173 ± 46 nM was determined. The Kd value was confirmed by ligand depletion analysis and equilibrium state measurements. Applying increasing concentrations of cAMP led to a concentration-dependent decrease in fluorescence intensity, indicating a displacement of 8-Fluo-cAMP. A Ki-value of 8.5 ± 2 µM was determined. The linear relationship of IC50 values obtained for cAMP as a function of ligand concentration confirmed the competitive binding mode: IC50: 13 ± 2 µM/16 ± 3 µM/23 ± 1 µM/27 ± 1 µM for 50 nM/150 nM/250 nM/500 nM 8-Fluo-cAMP. A similar competitive mode of binding was confirmed for 7-CH-cAMP, and an IC50 value of 230 ± 41 nM and a Ki of 159 ± 29 nM were determined. Two established drugs were tested in the assay. Ivabradine, an approved HCN channel pore blocker and gabapentin, is known to bind to HCN4 channels in preference to other isoforms with an unknown mode of action. As expected, ivabradine had no impact on ligand binding. In addition, gabapentin had no influence on 8-Fluo-cAMP's binding to HCN4-CNBD. This is the first indication that gabapentin is not interacting with this part of the HCN4 channel. The ligand-binding assay as described can be used to determine binding constants for ligands such as cAMP and derivatives. It could also be applied for the identification of new ligands binding to the HCN4-CNBD.

Dysfunctions of the C-type natriuretic peptide signaling pathway involved in the development of thoracic aortic aneurysm in Marfan patients

Marfan Syndrome (MFS) is an autosomal dominant inherited connective tissue disorder affecting the cardio-vascular system. Development of thoracic aortic aneurysms (TAA) leading to dissections and ruptures is the primary cause of morbidity and mortality in these patients. No treatment really cures Marfan patients. Interestingly, in humans, mutations in the gene coding for the C-type natriuretic peptide (CNP) or its receptors lead to a “Marfan-like syndrome”. CNP is a local regulator of skeletal growth and of vascular homeostasis, remodeling and angiogenesis. CNP binds to NPR-B or NPR-C receptors, both expressed on endothelial and vascular smooth muscle cells (vSMCs) and modulates the intracellular cGMP and cAMP levels. The aim of this project is to determine whether altered CNP signaling pathway contributes to TAA development and progression to rupture in Marfan patients. Plasma and aorta biopsies were taken from Marfan patients and from Fbn1C1039G/+ mice. The CNP, TGF-beta, cAMP and cGMP concentrations were measured in the plasma. mRNA and protein levels evaluated by qRT-PCR, and Western blot analysis in aortic tissue. Male and female mice studied and alterations of the aorta structure analyzed by ultrasound imaging. Plasma proCNP level in Marfan patients depends on the age and the TAA development. Increased levels found in young patients presenting no or reduced TAA and decreased levels in patients with TAA compared to age and sex-matched non-Marfan patients. CNP and NPR-C decreased at the mRNA and protein levels in the aorta of Marfan patients when compared to the same aorta in non-Marfan patients. In Fbn1+/- mice, aortic structure alterations are detected from 10 weeks in males and 34 weeks in females. CNP and NPR-C mRNA and protein levels drastically decreased in the thoracic aorta in 2–7 week-old male mice and in 34 week-old female mice. In 52-week old Fbn1+/- mice, the plasma cAMP level is increased in males and not in females, whereas the plasma cGMP protein level is increased in females but not in males. CNP injections in 3-week old Fbn1+/- male mice partially prevented the aorta structure alterations. A deficit in CNP protein level or in the expression of the NPR-C receptor contributes to the development and progression of TAA.

A cyclic effect of cAMP and calcium signaling contributes to jujube growth and development

3′,5′-Cyclic adenosine monophosphate (cAMP) is an important metabolite that is specifically enriched in jujube. However, the effect of cAMP on jujube cellular responses has not been comprehensively studied. Here, we established jujube cell suspension cultures and investigated the calcium influx in response to cAMP treatment through protoplast isolation and fluorescence intensity. Firstly, cAMP treatment could promote jujube growth and increase the content of endogenous cAMP. Using transcriptome analysis with transgenic Arabidopsis plants overexpressing adenylate cyclase (ZjAC) as a positive control, we identified 60 calcium-related differential expressed genes (DEGs) that contributed to the calcium signaling and inter- or intra-cellular responses. Pharmacological treatments such as cAMP and the calcium ionophore A23187 could induce ZjAC expression, the accumulation of cAMP and calcium influx in jujube cells, while ethylene glycol tetraacetic acid (EGTA) or bithionol treatment inhibited these changes. Moreover, the calcium channels and transporters in calcium influx, such as the ZjCNGC2 channel and the mitogen activated protein (MAP) kinase pathway, could be activated by cAMP treatment. In summary, our findings demonstrated that cAMP biosynthesis is dependent on calcium influx and the amplifying effect between calcium and cAMP may be involved in intracellular signal induction, which might contribute to the growth and development of jujube.

Mitochondria-Stimulating and Antioxidant Effects of Slovak Propolis Varieties on Bovine Spermatozoa

Propolis is a natural honeybee product, which is known for its beneficial pharmacological effects and use in traditional medicine. This study aspired to investigate the chemical and antioxidant properties of five propolis specimens, followed by the analysis of their in vitro impact on bovine spermatozoa vitality and oxidative profile. Semen samples from 10 breeding bulls were incubated in the absence or presence of 50 µg/mL propolis extracts, and selected sperm quality parameters including motility, mitochondrial activity, cAMP concentration, NAD+/NADH ratio, reactive oxygen species (ROS) and superoxide production were assessed at 2 h and 24 h. Sperm motion behavior and mitochondrial function were stabilized particularly in the presence of propolis collected from Hrabské and Litpovský Hrádok, Slovakia. At the same time, all propolis specimens acted as significant ROS quenchers and stabilized the oxidative milieu of the sperm cultures. Our data suggest that propolis may stabilize the mitochondrial function of spermatozoa while preventing excessive oxidative insults, thereby showing the potential to sustain the sperm motility of extended semen.

Midazolam nanocrystal facilitates submandibular lymphatic metabolism via cAMP signal transduction pathway

Midazolam is a short-acting benzodiazepine for sedation, but its application is limited by its poor solubility. This study explored the impact of midazolam nanocrystals on Cyclic adenosine monophosphate (cAMP0 signal transduction. A rat model of hypercalcemia was established and divided into model group, control group, midazolam nanocrystal group, midazolam group, inhibitor group and agonist group. After treatment, the contents of cAMP and cGMP were detected by radioimmunoassay, and transformation rate, proliferation and apoptosis of lymphocytes were analyzed. Additionally, we observed microcirculation of lymphocytes and calculated contraction activity, overall lymphatic contractile activity and lymph dynamic index. The structure of the prepared nanocrystals was uniform and stable. Higher cGMP concentration and lymph viscosity of rats in the midazolam nanocrystal, midazolam and inhibitor groups were lower, while cGMP was raised in the agonist group. Besides, modeled rats exhibited decreased cAMP concentration, T lymphocyte conversion rate, lymphatic vessel contraction frequency, contraction activity index, total contraction activity index, lymph dynamic index/flow and lymphocyte output (p &lt; 0.05), which were all increased in the midazolam nanocrystal group, midazolam group and agonist group but decreased in the inhibitor group (p &lt; 0.05). Moreover, Midazolam nanocrystals improved lymphocyte proliferation and decreased apoptosis (&lt;0.05). Midazolam nanocrystals can increase the concentration of cAMP and conversion rate of T lymphocytes, thereby reducing the concentration of cGMP, promoting lymphocyte proliferation, inhibiting apoptosis, and effectively improving the contraction frequency and contraction activity of lymphatic vessels. The cGMP can elevate total contractile activity index and lymph dynamics index, and enhance lymphatic transport function, while reducing lymph viscosity.