Search Results (1,153)

Vascular smooth muscle cells (VSMCs), the contractile cells in the tunica media of blood vessels, maintain vascular tone. The proliferation of VSMCs is an important feature of vascular remodeling that contributes to the regulation of blood pressure. Autophagy, an intracellular self-degrading process that delivers cytoplasmic constituents to lysosomes, plays a vital role in VSMC proliferation. This is regulated by the dopaminergic and renin–angiotensin systems but their interplay in their regulation of autophagy in VSMCs is not well-understood. In rat VSMCs, fenoldopam (Fen), a dopamine D1-like receptor agonist, increased autophagy, as determined by the increase in the protein expressions of microtubule-associated protein 1 light chain (LC)3-II and beclin-1 (BECN1), in a time- and concentration-dependent manner. Conversely, angiotensin II (Ang II), the endogenous Ang II type 1 receptor (AT₁R) agonist, decreased the protein expression of LC3-II and BECN1, also in a time- and concentration-dependent manner. The production of cyclic adenosine monophosphate (cAMP) and autophagic LC3-II puncta in VSMCs were increased by Fen and decreased by Ang II. Pre-treatment of VSMCs with Rp-cAMPS, a protein kinase A inhibitor, prevented the Fen-mediated increase and the Ang II-mediated decrease in LC3-II protein expression. Fen decreased, whereas Ang II increased the phosphorylation of P70S6K, a direct downstream mammalian target of rapamycin (mTOR). The inhibitory effect of Fen and stimulatory effect of Ang II on P70S6K phosphorylation were prevented by Rp-cAMPS. Ang II also decreased the Fen-mediated increase in cAMP production, while Fen attenuated the Ang II-mediated increase in cell proliferation, a response that occurs downstream of autophagy. Moreover, Ang II prevented the Fen-mediated inhibition of cell proliferation, an effect that was blocked by losartan, an AT₁R antagonist. These results demonstrate that Fen and Ang II counter-regulate autophagy and proliferation of VSMCs via the mTOR pathway, which is cAMP-dependent. Full article

Adenylate cyclase 5 (Adcy5) generates cyclic adenosine monophosphate (cAMP) downstream of G protein-coupled receptor signaling, yet its role in vertebrate pigmentation remains incompletely understood. Here, we generated a CRISPR/Cas9-mediated adcy5 knockout line in Nile tilapia (Oreochromis niloticus) to investigate its function in chromatophore biology. Loss of adcy5 resulted in a pronounced disruption of body coloration, characterized by the absence of vertical black bars and a global reduction in pigmentation. Despite this, chromatophore number was largely unaffected, indicating that Adcy5 is not required for pigment cell specification but is essential for functional pigmentation. At the cellular level, pigment granules in melanophores and erythrophores failed to undergo dispersion and instead remained constitutively aggregated, revealing a primary defect in intracellular pigment granule transport. Consistently, adcy5 mutants exhibited reduced expression of key melanogenesis-associated genes, including mitfa, tyrb, tyrp1a, and tyrp1b, accompanied by decreased melanin content across multiple tissues. Pharmacological activation of cAMP signaling partially rescued the pigment dispersion defect, whereas stimulation of upstream α-MSH signaling produced only limited effects, placing Adcy5 upstream of intracellular cAMP production within the pigment regulatory cascade. Importantly, we further demonstrate that Adcy5 is required for erythrophore pigment granule dispersion, extending its functional role beyond melanophore biology. Together, these findings identify Adcy5 as a conserved regulator integrating cAMP-dependent pigment synthesis and granule transport across multiple chromatophore types in teleost fish. Full article

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a desmoplastic, immunosuppressive tumor microenvironment enriched in extracellular adenosine. High adenosine levels disrupt anti-tumor immunity by engaging adenosine receptors on T cells, macrophages, and dendritic cells. In this context, strategies to reduce extracellular adenosine signaling within the TME are in focus. Among them, enzymatic degradation of extracellular adenosine levels has emerged as a promising approach to overcome immune suppression and restore anti-tumor immune responses. Methods: Here, we engineered an E. coli strain to express a surface-bound form of a bacterial ADA2 enzyme and used the outer membrane vesicles produced by this strain as nanoparticles carrying the functional enzyme. Our data indicate robust surface expression and enzymatic activity of ADA2 on OMV particles. Results: In a PDAC subcutaneous flank tumor model, intratumoral administration of OMV-ADA2 particles was associated with significantly increased infiltration of CD8⁺ T cells and Granzyme B expression and concomitant decreases in collagen deposition and α-smooth muscle actin (α-SMA) positive stromal cells, suggesting modulation of the desmoplastic stroma. Conclusions: These findings support further investigation of OMV–bADA2 as a potential platform to modulate tumor metabolism and stroma, activate anti-tumor immunity, and serve as a carrier for co-delivery of additional anticancer modalities in PDAC. Full article

The endocannabinoid system (ECS) is increasingly recognized as a central regulator of immune homeostasis, integrating neural, metabolic, and immune signaling to maintain physiological equilibrium. This Perspective examines the “therapeutic paradox” of endocannabinoid immunomodulation, whereby anti-inflammatory and tissue-protective effects are mechanistically linked to transient immunosuppression. Although cannabinoid receptor 2 (CB2) is the primary mediator of immune regulation, growing evidence indicates that cannabinoid receptor 1 (CB1) also contributes to inflammatory control in both the central nervous system and peripheral tissues. Activation of CB2 suppresses inflammatory signaling through Gi/o-mediated inhibition of adenylate cyclase, reduced cyclic adenosine monophosphate (cAMP) signaling, and repression of nuclear factor kappa B (NF-κB)-dependent transcription. While these mechanisms limit pathological inflammation and promote tissue protection, they simultaneously attenuate innate and adaptive immune functions required for effective pathogen clearance. Across neuroinflammatory disorders, inflammatory bowel disease, hepatic injury, sepsis, cancer, and systemic inflammatory syndromes, the ECS shifts immune responses toward resolution at the cost of reduced antimicrobial readiness. We synthesize the molecular mechanisms underlying this therapeutic paradox, including macrophage polarization, lymphocyte reprogramming, and tissue-specific immune adaptations, and discuss strategies for developing endocannabinoid-based therapeutics that preserve anti-inflammatory efficacy while minimizing immunosuppressive liabilities. Full article

Background and Objectives: Neuropathic pain presents a significant therapeutic challenge, often due to resistance to opioids. This study examined how selective adenosine receptor agonists and allopurinol modulate spinal mu-opioid receptor (MOR) mRNA expression in a rat model of neuropathic pain, aiming to develop a novel strategy for restoring opioid homeostasis. Materials and Methods: Male Sprague-Dawley rats were subjected to L5 spinal nerve ligation (SNL). The animals received treatment with selective A1AR (CCPA, 1 mg/kg) or A3AR (IB-MECA, 1 mg/kg) agonists, or allopurinol (10 or 50 mg/kg) for three days. Spinal MOR mRNA expression was measured on day 7 post-SNL using qRT-PCR. Results: SNL resulted in a non-significant upward trend in MOR transcription. Selective agonists (CCPA and IB-MECA) appeared to enhance or maintain MOR levels compared to the vehicle-treated group, but the most pronounced trend toward recovery of MOR mRNA expression was observed with high-dose allopurinol (50 mg/kg). Effect size analysis revealed large Cohen’s d values for high-dose allopurinol (d = 1.05) and CCPA (d = 0.90) versus sham, supporting the biological relevance of these preliminary trends. Conclusions: These preliminary findings suggest that augmenting endogenous adenosine bioavailability with allopurinol may be more effective than targeting individual receptor subtypes in modulating the spinal opioid system. As this is an exploratory pilot investigation, these results should be interpreted as hypothesis-generating rather than confirmatory. Since allopurinol is already a clinically established drug, these findings offer a mechanistic rationale warranting further investigation of its potential as an adjuvant for opioid resistance in chronic pain management. Full article

As a natural purine metabolite, inosine’s impact on kidney-related diarrhea through its influence on gut microbiota and associated metabolic functions remains unclear. Kidney-related diarrhea was induced in male KM mice. Histopathological alterations and inflammatory infiltration were assessed using hematoxylin and eosin (HE) staining. ELISA was used to measure corticosterone (CORT), antidiuretic hormone (ADH), and adenosine triphosphate (ATP) to assess metabolic status and fluid homeostasis. Immunohistochemistry (IHC) staining techniques, Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), and Western blot (WB) were used to analyze the expression of aquaporin-4 (AQP4), AMP-activated protein kinase (AMPK), nuclear factor kappa-B (NF-κB), and adenosine A2A Receptor (A2AR). Gut microbiota composition and predicted functional pathways were analyzed using 16S rRNA sequencing and KEGG-based functional prediction, followed by correlation analyses between the microbiota and factors. Inosine improved renal function, alleviated renal and colonic histopathological damage, and reduced inflammatory infiltration. It also increased ATP, ADH, and CORT levels, indicating improvements in metabolic and fluid-balance-related factors. Inosine also increased A2AR, AMPK, and AQP4 expression while decreasing NF-κB expression. Moreover, inosine altered gut microbial composition and was associated with differences in predicted microbial functional pathways. Significant correlations were observed between specific bacterial taxa and host indicators. Inosine alleviated kidney-related diarrhea in mice, accompanied by improvements in metabolic status and fluid homeostasis, reduced inflammation, modulation of A2AR/AMPK/NF-κB-related signaling pathway, and alterations in gut microbial composition. Further studies are required to clarify the causal contributions of gut microbial composition and functional activity to the beneficial effects of inosine. Full article

Acute kidney injury (AKI) is characterized by a rapid decline or sudden loss of renal function over hours to days. Pathophysiological triggers such as renal ischemia–reperfusion (IR) injury and the accumulation of uremic toxins (UTs), notably indoxyl sulfate (IS), can initiate AKI and affect vascular beds distant from the ischemic site, like the aorta. In this context, purinergic signaling becomes relevant, since its components regulate vascular tone and inflammatory responses. This study aimed to evaluate the impact of AKI induced by IR with or without IS administration on purinergic signaling in the aorta of mice. Renal ischemia was induced by the occlusion of the left renal pedicle for 60 min, followed by reperfusion for 8 days (IR 8) or 15 days (IR 15). Some animals were also treated with saline solution or IS for 15 days. The IR15 group exhibited increased plasma IS concentrations and upregulated adenosine receptor gene expression. Furthermore, in the IR+IS group, there was increased expression of A1, A2a, NTPDase 1, and 2. This shift toward an adenosine-enriched signaling environment may represent a key mechanism linking renal injury to systemic vascular inflammation. Full article

Major depressive disorder (MDD) involves dysregulation of the hypothalamic–pituitary–adrenal axis, neuroinflammation, serotonergic dysfunction, and impaired neurotrophic signaling. Whether combining corticosterone (CORT) with chronic restraint stress (CRS) produces a more comprehensive depression-like phenotype than either model alone remains unexplored. Male C57BL/6N mice were assigned to four groups—Sham (n = 7), CORT (20 mg/kg s.c.; n = 7), CRS (3 h/day; n = 7), and CORT + CRS (C+C; n = 8)—and evaluated by behavioral tests, hippocampal qRT-PCR, Western blot, LC-MS/MS metabolomics, and immunohistochemistry. All experimental groups showed elevated immobility in tail suspension and forced swim tests without inter-group differences. Two-way ANOVA revealed a behavioral–molecular dissociation: FST immobility showed CORT and CRS main effects without a significant interaction, whereas hippocampal LC-MS/MS analytes exhibited strong CORT × CRS interactions. The C+C group showed the strongest 5-HT1A receptor (HTR1A) upregulation, the greatest reductions in Trk-b and DCX, and unique decreases in serum dopamine, glutamine, and adenosine. Representative Western blot densitometry indicated the largest p-ERK/ERK reduction in C+C (54%), while p-CREB/CREB was most suppressed in CRS (52%). BDNF and NeuN proteins were lowest in C+C (68% and 61% reductions). Combined CORT + CRS produces the most comprehensive depression-related molecular profile, integrating multiple pathological domains. Full article

Pemafibrate (PEM), a novel selective peroxisome proliferator-activated receptor α modulator, is widely used to treat dyslipidemia, yet its systemic metabolic effects remain incompletely defined. We performed untargeted serum metabolomics in patients with hypertriglyceridemia at baseline and at 2 and 8 weeks after PEM treatment. PEM increased cystine, L-methionine, uridine, and L-carnitine, while decreasing lipid-related metabolites, including lysophosphatidylcholines, adenosine, and erucic acid (22:1). Circulating carnitine levels rose progressively, with a significant elevation at 8 weeks, whereas ketone bodies showed only modest, non-significant increases. Consistently, increases in circulating and tissue carnitine were also observed in male 8-week-old C57BL/6J mice treated with a clinically relevant dose of PEM for 2 weeks. Mechanistically, PEM did not significantly alter genes involved in carnitine biosynthesis or transport, but upregulated hepatic carnitine O-acetyltransferase and carnitine O-octanoyltransferase, key enzymes of carnitine utilization and turnover. Collectively, these findings suggest that enhanced carnitine metabolism represents an important metabolic axis of PEM action. Full article

Intramuscular fat content is a key determinant of pork quality, influencing traits such as tenderness, juiciness, and flavor. However, the molecular mechanisms regulating intramuscular fat deposition in indigenous pig breeds remain incompletely understood. This study aimed to identify genes and regulatory mechanisms associated with intramuscular fat accumulation in Hezuo pigs. Longissimus dorsi muscle samples from Hezuo pigs with extreme high and low intramuscular fat contents were subjected to chromatin accessibility profiling and transcriptome sequencing. Comparative analyses identified 2201 differentially accessible chromatin regions and 588 differentially expressed genes between the two groups. Functional enrichment analyses indicated that these genes were mainly involved in lipid metabolism, focal adhesion, extracellular matrix–receptor interaction, fatty acid metabolism, and adenosine monophosphate-activated protein kinase signaling. Integration of chromatin accessibility and gene expression datasets identified 92 co-regulated genes associated with intramuscular fat deposition, including MYLK3, PDGFC, PAK1, IGF1R, LAMA4, DIAPH1, SDC4, GADD45G, and RXRG. These findings reveal regulatory networks underlying intramuscular fat accumulation in Hezuo pigs and provide candidate genes and molecular resources for improving meat quality through genetic selection and breeding programs. Full article

Brain injury after hypoxia–ischemia (HI) is the leading cause of morbidity and mortality in term and near-term neonates worldwide. The ferret is a promising translational model to study HI due to its gyrified brain and white-to-gray matter ratio that more closely resembles humans compared to rodents. Caffeine, an adenosine A2A receptor (A2AR) antagonist, shows neuroprotective potential after HI, but its effects have not been fully characterized. We sought to evaluate caffeine’s effect on neuronal cell death, cytotoxicity, and inflammatory and oxidative stress markers in a term-equivalent ferret organotypic brain slice model of HI. Slices were cultured for 72 h, exposed to two hours of oxygen–glucose deprivation (OGD), and randomized to OGD alone, OGD with caffeine (20 or 50 mg/L), or OGD with caffeine and an A2AR agonist. Healthy slices served as controls. Outcomes included global cell death, regional cell death, microglial morphology, and expression of inflammatory and oxidative stress genes (46–48 slices/group for cell death assays and 18 slices/group for imaging, balanced by sex). Caffeine 50 mg/L significantly reduced global cell death compared to OGD (p = 0.02), and this effect persisted despite co-administration of an A2AR agonist (p = 0.01), suggesting that protection was not primarily mediated through A2AR signaling. Caffeine also did not change regional pyknotic nuclei counts (p > 0.05). Caffeine altered microglial morphology, increasing the proportion of microglia with features characteristic of control conditions. OGD significantly increased expression of inflammatory and oxidative stress-related genes (p < 0.05) compared with control slices, whereas caffeine did not significantly alter gene expression. In summary, caffeine partially reversed global cell death after OGD and altered microglial morphology. Larger, higher-powered studies are needed to further investigate caffeine’s effects on neonatal HI. Full article

Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death in American men. Most patients with metastatic disease respond initially to androgen deprivation therapy (ADT) but almost inevitably progress to castration-resistant prostate cancer (CRPC). Identification of markers and drivers of Metastatic CRPC (mCRPC) that (a) represent a progenitor-type cancer cell population, (b) persist in castration-resistant disease, (c) are actionable targets expressed on the cell surface, and (d) are induced by hypoxia is required to facilitate the development of novel targeted therapies. We identified prostatic acid phosphatase (PAP), particularly the transmembrane form (TMPAP), as one such potential target. PAP is both a phosphatase and a 5′ectonucleotidase that generates adenosine. PAP is a human tumor marker first described in 1936 and is still used as an important prognostic marker for advanced metastatic prostate cancer. Our group recently reported that the transmembrane form of the protein (TMPAP) is expressed in CRPC and can serve as a potential therapeutic target. We identified a lead human anti-TMPAP antibody clone 3D8 (3D8-Ab). 3D8-ADCs (Antibody Drug Conjugates) and 3D8-Ab were tested for their ability to reduce tumor size/volume in a xenograft model. The human PAP-expressing PCa cell line VCaP, originally derived from a vertebral metastasis from a patient with CRPC, was inoculated subcutaneously into SCID mice. Treatment with either 3D8-Ab or 3D8-ADC significantly reduced tumor size and increased animal survival. These data indicate that targeting PAP with monoclonal antibodies either alone or conjugated to toxins has the potential to treat CRPC. Full article

Caffeine is among the most widely used supplements in sports nutrition, with substantial evidence supporting its efficacy for enhancing athletic performance. However, the relevance of pre-exercise caffeine supplementation extends beyond its ergogenic effects. Exercise itself acts as an acute cardiometabolic stressor, eliciting dynamic responses in blood pressure, heart rate, vascular tone, substrate utilization, and glucose regulation. Caffeine may further modify the magnitude, temporal profile, and recovery kinetics of these responses. This focused narrative review examines the acute cardiometabolic responses associated with pre-exercise caffeine supplementation and its potential regulatory effects. It outlines the underlying biological mechanisms, including adenosine receptor antagonism, hemodynamic and vascular pathways, and metabolic and substrate-utilization pathways, while synthesizing current evidence from the literature. Available evidence indicates that the acute effects of caffeine are highly context dependent. In some exercise settings, caffeine may promote lipolysis and fat oxidation; in others, particularly among susceptible individuals or under specific exercise conditions, it may be associated with greater acute cardiovascular or autonomic load, reflected by higher peripheral vascular resistance and blood pressure, altered vascular reactivity, or delayed post-exercise autonomic recovery. Its effects may vary according to dose, timing, supplement form, exercise modality and intensity, training status, habitual caffeine intake, genotype, sex, and hormonal status. Overall, pre-exercise caffeine should not be regarded solely as a uniformly beneficial ergogenic aid, but rather as a physiological modulator that may reshape the acute cardiometabolic milieu during and after exercise. Future studies should integrate hemodynamic, vascular, metabolic, and individual-variability measures over extended observation periods to support more evidence-based and individualized guidance on the appropriate use of pre-exercise caffeine. Full article

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger downstream of G protein-coupled receptors (GPCRs) and a central regulator of cellular signaling. Genetically encoded exchange proteins directly activated by cAMP (Epac)-based Förster resonance energy transfer (FRET) biosensors enable real-time monitoring of cAMP dynamics in living cells, but commonly used cyan/yellow FRET pairs require short-wavelength excitation, limiting compatibility with multiplex imaging and blue-light optogenetic tools such as bacterial photoactivated adenylyl cyclases (bPACs). Here, we engineered and systematically characterized four red-shifted Epac-based single-chain FRET cAMP sensors combining yellow or orange FRET donors with red fluorescent FRET acceptors. Using ratiometric live-cell imaging, we quantified stimulus-evoked FRET responses and identified Epac_red4 as the best-performing variant, showing an approximately 55% decrease in normalized FRET after forskolin stimulation. Epac_red4 also reliably detected G_i/o-mediated decreases in cAMP following μ-opioid receptor activation. Brief 405 nm light pulses induced graded and reversible cAMP elevations using the low dark-activity variant bPAC-F198Y. Furthermore, Epac_red4 enabled analysis of cAMP recovery kinetics during phosphodiesterase inhibition and multiplex imaging of cAMP and intracellular Ca²⁺ using Fura-2 with minimal spectral and pH-related interference under physiological imaging conditions. Together, Epac_red4 represents a robust red-shifted cAMP sensor for optogenetic and multiplex signaling studies. Full article

Fibrosis is a common extracellular matrix pathology characterized by increased scarring, representing a critical checkpoint toward cirrhosis and hepatocellular carcinoma. Its onset involves coordinated interplay among hepatocytes, Kupffer, and hepatic stellate cells (HSCs). Extracellular ATP and its derivates act as crucial damage-associated molecular patterns when released by injured liver cells, binding to specific purinergic receptors (P2X, P2Y, and P1) to establish an autocrine–paracrine signaling loop. The hepatic fibrotic response underlies the activation of ATP receptors that generate second messengers and cationic conductance. In parallel, extracellular nucleotidases hydrolyze ATP towards less phosphorylated intermediates and adenosine. This review focuses on the role of P2X and P2Y receptors in liver injury. The P2X7 receptor regulates the NLRP3 inflammasome in Kupffer cells and HSCs, while the P2X4 receptor is upregulated in myofibroblasts, modulating migration and matrix synthesis. Among P2Y receptors, P2Y2 drives inflammation and steatosis but promotes HIF-1α-mediated DNA repair. The P2Y6 receptor promotes alcohol-induced injury but restrains metabolic-dysfunction-associated steatohepatitis. P2Y2 and P2Y4 receptors maintain biliary homeostasis in cholangiocytes, whereas the P2Y1 receptor preserves HSC quiescence by blocking YAP translocation. Finally, UDP-glucose–P2Y14 induces HSC activation. Targeting these specific purinergic receptors or ecto-nucleotidases represents a promising pharmacological frontier against hepatic fibrosis. Full article