Modelling and analysis of cAMP-induced mixed-mode oscillations in cortical neurons: Critical roles of HCN and M-type potassium channels.

Cyclic AMP controls neuronal ion channel activity. For example hyperpolarization-activated cyclic nucleotide-gated (HCN) and M-type K+ channels are activated by cAMP. These effects have been suggested to be involved in astrocyte control of neuronal activity, for example, by controlling the action potential firing frequency. In cortical neurons, cAMP can induce mixed-mode oscillations (MMOs) consisting of small-amplitude, subthreshold oscillations separating complete action potentials, which lowers the firing frequency greatly. We extend a model of neuronal activity by including HCN and M channels, and show that it can reproduce a series of experimental results under various conditions involving and inferring with cAMP-induced activation of HCN and M channels. In particular, we find that the model can exhibit MMOs as found experimentally, and argue that both HCN and M channels are crucial for reproducing these patterns. To understand how M and HCN channels contribute to produce MMOs, we exploit the fact that the model is a three-time scale dynamical system with one fast, two slow, and two super-slow variables. We show that the MMO mechanism does not rely on the super-slow dynamics of HCN and M channel gating variables, since the model is able to produce MMOs even when HCN and M channel activity is kept constant. In other words, the cAMP-induced increase in the average activity of HCN and M channels allows MMOs to be produced by the slow-fast subsystem alone. We show that the slow-fast subsystem MMOs are due to a folded node singularity, a geometrical structure well known to be involved in the generation of MMOs in slow-fast systems. Besides raising new mathematical questions for multiple-timescale systems, our work is a starting point for future research on how cAMP signalling, for example resulting from interactions between neurons and glial cells, affects neuronal activity via HCN and M channels.

Ginkgetin effectively mitigates collagen and AA-induced platelet activation via PLCγ2 but not cyclic nucleotide-dependent pathway in human.

Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 µM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca2+ ]i) levels and thromboxane A2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoproteinSer157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs.

Co-delivery of doxorubicin and STING agonist cGAMP for enhanced antitumor immunity.

Many chemotherapeutic agents can induce immunogenic cell death (ICD), which leads to the release of danger-associated molecular patterns (DAMPs) and tumor-associated antigens. This process promotes dendritic cells (DCs) maturation and cytotoxic T lymphocyte (CTL) infiltration. However, cancer cells can employ diverse mechanisms to evade the host immune system. Recent studies have shown that stimulator of interferon genes (STING) agonists, such as cGAMP, can amplify ICD-triggered immune responses and enhance the infiltration of immune cells into the tumor microenvironment (TME). Building upon these findings, we constructed a doxorubicin (DOX) and cGAMP co-delivery system (DOX/cGAMP@NPs) for melanoma and triple-negative breast cancer (TNBC) therapy. The results demonstrated that DOX could effectively destroy tumors and induce the release of DAMPs by ICD. Furthermore, in orthotopic 4T1 tumors mice model and subcutaneous B16 tumor mice model, cGAMP could promote the maturation of DCs and CD8+ T cell activation and infiltration by inducing the secretion of type I interferons and pro-inflammation cytokine, which amplified the antitumor immune response induced by DOX. This strategy also promoted the depletion of immunosuppressive cells, potentially alleviating the immunosuppressive TME. In conclusion, our study highlights the combination of DOX-induced ICD and the immune-enhancing properties of cGAMP holds significant implications for future research and clinical applications.

Calcium-sensing receptor activator cinacalcet for treatment of cyclic nucleotide-mediated secretory diarrheas.

Cyclic nucleotide elevation in intestinal epithelial cells is the key pathology causing intestinal fluid loss in secretory diarrheas such as cholera. Current secretory diarrhea treatment is primarily supportive, and oral rehydration solution is the mainstay of cholera treatment. There is an unmet need for safe, simple and effective diarrhea treatments. By promoting cAMP hydrolysis, extracellular calcium-sensing receptor (CaSR) is a regulator of intestinal fluid transport. We studied the antidiarrheal mechanisms of FDA-approved CaSR activator cinacalcet and tested its efficacy in clinically relevant human cell, mouse and intestinal organoid models of secretory diarrhea. By using selective inhibitors, we found that cAMP agonists-induced secretory short-circuit currents (Isc) in human intestinal T84 cells are mediated by collective actions of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) and Clc-2 Cl- channels, and basolateral membrane K+ channels. 30 µM cinacalcet pretreatment inhibited all 3 components of forskolin and cholera toxin-induced secretory Isc by ∼75%. In mouse jejunal mucosa, cinacalcet inhibited forskolin-induced secretory Isc by ∼60% in wild type mice, with no antisecretory effect in intestinal epithelia-specific Casr knockout mice (Casr-flox; Vil1-cre). In suckling mouse model of cholera induced by oral cholera toxin, single dose (30 mg/kg) oral cinacalcet treatment reduced intestinal fluid accumulation by ∼55% at 20 hours. Lastly, cinacalcet inhibited forskolin-induced secretory Isc by ∼75% in human colonic and ileal organoids. Our findings suggest that CaSR activator cinacalcet has antidiarrheal efficacy in distinct human cell, organoid and mouse models of secretory diarrhea. Considering its excellent clinical safety profile, cinacalcet can be repurposed as a treatment for cyclic nucleotide-mediated secretory diarrheas including cholera.

Vinylphosphonate-based cyclic dinucleotides enhance STING-mediated cancer immunotherapy.

Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.

Modulating cyclic nucleotides pathways by bioactive compounds in combatting anxiety and depression disorders.

Anxiety and depression disorders are highly prevalent neurological disorders (NDs) that impact up to one in three individuals during their lifetime. Addressing these disorders requires reducing their frequency and impact, understanding molecular causes, implementing prevention strategies, and improving treatments. Cyclic nucleotide monophosphates (cNMPs) like cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), cyclic uridine monophosphate (cUMP), and cyclic cytidine monophosphate (cCMP) regulate the transcription of genes involved in neurotransmitters and neurological functions. Evidence suggests that cNMP pathways, including cAMP/cGMP, cAMP response element binding protein (CREB), and Protein kinase A (PKA), play a role in the physiopathology of anxiety and depression disorders. Plant and mushroom-based compounds have been used in traditional and modern medicine due to their beneficial properties. Bioactive compound metabolism can activate key pathways and yield pharmacological outcomes. This review focuses on the molecular mechanisms of bioactive compounds from plants and mushrooms in modulating cNMP pathways. Understanding these processes will support current treatments and aid in the development of novel approaches to reduce the prevalence of anxiety and depression disorders, contributing to improved outcomes and the prevention of associated complications.

Roflumilast Protects against Neuroinflammatory Alterations in Brain Tissues of Lipopolysaccharide-induced Mice Model.

BACKGROUND: Microglial overactivation promotes the production of various second messengers and inflammatory markers in brain tissue, resulting in neuroinflammation and neurodegeneration, which may lead to cognitive decline. The cyclic nucleotides are one of the important second messengers involved in the regulation of neurogenesis, synaptic plasticity, and cognition. The levels of these cyclic nucleotides are maintained by phosphodiesterase enzyme isoforms, particularly PDE4B, in the brain. An imbalance between PDE4B levels and cyclic nucleotides may lead to aggravating neuroinflammation. METHODS: Lipopolysaccharides (LPS) were administered intraperitoneally on alternate days for 7 days at a dose of 500 µg/kg in mice, which triggered systemic inflammation. This may lead to the activation of glial cells and may activate oxidative stress and neuroinflammatory markers in brain tissue. Furthermore, oral administration of roflumilast (0.1, 0.2, and 0.4 mg/kg) in this model ameliorated oxidative stress markers, neuroinflammation and improved neurobehavioral parameters in these animals. RESULTS: The detrimental effect of LPS increased oxidative stress, AChE enzyme levels, and decreased catalase levels in brain tissues, along with memory impairment in animals. Moreover, it also enhanced the activity and expression of the PDE4B enzyme, resulting in a decline in cyclic nucleotide levels. Furthermore, treatment with roflumilast improved the cognitive decline, decreased AChE enzyme level, and increased the catalase enzyme level. Roflumilast also reduced the PDE4B expression in a dose-dependent manner, which LPS up-regulated. CONCLUSION: Roflumilast has shown an anti-neuroinflammatory effect and reversed the cognitive decline in LPS-induced mice model.

Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel.

Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.

ESCRT-dependent STING degradation inhibits steady-state and cGAMP-induced signalling.

Stimulator of interferon genes (STING) is an intracellular sensor of cyclic di-nucleotides involved in the innate immune response against pathogen- or self-derived DNA. STING trafficking is tightly linked to its function, and its dysregulation can lead to disease. Here, we systematically characterize genes regulating STING trafficking and examine their impact on STING-mediated responses. Using proximity-ligation proteomics and genetic screens, we demonstrate that an endosomal sorting complex required for transport (ESCRT) complex containing HGS, VPS37A and UBAP1 promotes STING degradation, thereby terminating STING-mediated signaling. Mechanistically, STING oligomerization increases its ubiquitination by UBE2N, forming a platform for ESCRT recruitment at the endosome that terminates STING signaling via sorting in the lysosome. Finally, we show that expression of a UBAP1 mutant identified in patients with hereditary spastic paraplegia and associated with disrupted ESCRT function, increases steady-state STING-dependent type I IFN responses in healthy primary monocyte-derived dendritic cells and fibroblasts. Based on these findings, we propose that STING is subject to a tonic degradative flux and that the ESCRT complex acts as a homeostatic regulator of STING signaling.

Theta patterns of stimulation induce synaptic and intrinsic potentiation in O-LM interneurons.

Brain oscillations have long-lasting effects on synaptic and cellular properties. For instance, synaptic stimulation at theta (θ) frequency induces persistent depression of both excitatory synaptic transmission and intrinsic excitability in CA1 principal neurons. However, the incidence of θ activity on synaptic transmission and intrinsic excitability in hippocampal GABAergic interneurons is unclear. We report here the induction of both synaptic and intrinsic potentiation in oriens-lacunosum moleculare (O-LM) interneurons following stimulation of afferent glutamatergic inputs in the θ frequency range (∼5 Hz). Long-term synaptic potentiation (LTP) is induced by synaptic activation of calcium-permeable AMPA receptors (CP-AMPAR), whereas long-term potentiation of intrinsic excitability (LTP-IE) results from the mGluR1-dependent down-regulation of Kv7 voltage-dependent potassium channel and hyperpolarization activated and cyclic nucleotide-gated (HCN) channel through the depletion of phosphatidylinositol-4,5-biphosphate (PIP2). LTP and LTP-IE are reversible, demonstrating that both synaptic and intrinsic changes are bidirectional in O-LM cells. We conclude that synaptic activity at θ frequency induces both synaptic and intrinsic potentiation in O-LM interneurons, i.e., the opposite of what is typically seen in glutamatergic neurons.

Ivabradine treatment lowers blood pressure and promotes cardiac and renal protection in spontaneously hypertensive rats.

Hypertension is linked to hyperpolarization-activated cyclic nucleotide-gated (HCN) function, expressed in excitable and non-excitable cells. Considering that the reduction in heart rate (HR) improves coronary perfusion and cardiac performance, ivabradine (IVA) emerged as an important drug for the treatment of cardiovascular diseases. AIM: Evaluate if IVA chronic treatment effect can mitigate hypertension and reverse the cardiac and renal damage in SHR. MAIN METHODS: Rats were divided into 4 groups treated for 14 days with PBS (1 ml/kg; i.p) or IVA (1 mg/kg; i.p): 1) WKY PBS; 2) SHR PBS; 3) WKY IVA; and 4) SHR IVA. The systolic blood pressure (SBP) was measured, indirectly, before and during the treatment period with IVA (day 0, 1, 7 and 11). Rats were subjected to artery cannulation for direct blood pressure (BP) measurement. Morphofunctional and gene expression were evaluated in the heart and kidneys. KEY FINDINGS: IVA reduced SBP only in SHR on the 7th day. Direct blood pressure measurement showed that IVA chronic treatment reduced HR in the SHR. Interestingly, mean arterial pressure (MAP) was reduced in SHR IVA when compared to SHR PBS. Serum and urinary biochemical data were not altered by IVA. Moreover, IVA reduced the renal inflammatory infiltrates and increased glomerular density, besides preventing the cardiac inflammatory and hypertrophic responses. SIGNIFICANCE: IVA treatment lowered blood pressure, improved cardiac remodeling and inflammation, as well as decreasing renal damage in SHR. Further, IVA increased renal HCN2 mRNA and reduced cardiac HCN4 mRNA.

The NO/cGMP/PKG pathway in platelets: The therapeutic potential of PDE5 inhibitors in platelet disorders.

Platelets are the "guardians" of the blood circulatory system. At sites of vessel injury, they ensure hemostasis and promote immunity and vessel repair. However, their uncontrolled activation is one of the main drivers of thrombosis. To keep circulating platelets in a quiescent state, the endothelium releases platelet antagonists including nitric oxide (NO) that acts by stimulating the intracellular receptor guanylyl cyclase (GC). The latter produces the second messenger cyclic guanosine-3',5'-monophosphate (cGMP) that inhibits platelet activation by stimulating protein kinase G, which phosphorylates hundreds of intracellular targets. Intracellular cGMP pools are tightly regulated by a fine balance between GC and phosphodiesterases (PDEs) that are responsible for the hydrolysis of cyclic nucleotides. Phosphodiesterase type 5 (PDE5) is a cGMP-specific PDE, broadly expressed in most tissues in humans and rodents. In clinical practice, PDE5 inhibitors (PDE5i) are used as first-line therapy for erectile dysfunction, pulmonary artery hypertension, and lower urinary tract symptoms. However, several studies have shown that PDE5i may ameliorate the outcome of various other conditions, like heart failure and stroke. Interestingly, NO donors and cGMP analogs increase the capacity of anti-platelet drugs targeting the purinergic receptor type Y, subtype 12 (P2Y12) receptor to block platelet aggregation, and preclinical studies have shown that PDE5i inhibits platelet functions. This review summarizes the molecular mechanisms underlying the effect of PDE5i on platelet activation and aggregation focusing on the therapeutic potential of PDE5i in platelet disorders, and the outcomes of a combined therapy with PDE5i and NO donors to inhibit platelet activation.

Lanthanide-Nucleotide Coordination Nanoparticles for STING Activation.

Activation of the stimulator of interferon genes (STING) is essential for blocking viral infections and eliciting antitumor immune responses. Local injection of synthetic STING agonists, such as 2'3'-cGAMP [cGAMP = cyclic 5'-guanosine monophosphate (cGMP)-adenosine monophosphate (AMP)], is a promising approach to enhance antiviral functions and cancer immunotherapy. However, the application of such agonists has been hindered by complicated synthetic procedures, high doses, and unsatisfactory systemic immune responses. Herein, we report the design and synthesis of a series of 2'3'-cGAMP surrogates in nanoparticle formulations formed by reactions of AMP, GMP, and coordinating lanthanides. These nanoparticles can stimulate the type-I interferon (IFN) response in both mouse macrophages and human monocytes. We further demonstrate that the use of europium-based nanoparticles as STING-targeted adjuvants significantly promotes the maturation of mouse bone-marrow-derived dendritic cells and major histocompatibility complex class I antigen presentation. Dynamic molecular docking analysis revealed that these nanoparticles bind with high affinity to mouse STING and human STING. Compared with soluble ovalbumin (OVA), subcutaneously immunized europium-based nanovaccines exhibit significantly increased production of primary and secondary anti-OVA antibodies (∼180-fold) in serum, as well as IL-5 (∼28-fold), IFN-γ (∼27-fold), and IFN-α/ß (∼4-fold) in splenocytes ex vivo. Compared with the 2'3'-cGAMP/OVA formulation, subcutaneous administration of nanovaccines significantly inhibits B16F10-OVA tumor growth and prolongs the survival of tumor-bearing mice in both therapeutic and protective models. Given the rich supramolecular chemistry with lanthanides, this work will enable a readily accessible platform for potent humoral and cellular immunity while opening new avenues for cost-effective, highly efficient therapeutic delivery of STING agonists.

UV-B filter octylmethoxycinnamate-induced vascular endothelial disruption on rat aorta: In silico and in vitro approach.

Throughout human life, an extensive and varied range of emerging environmental contaminants, called endocrine disruptors (EDCs), cause adverse health effects, including in the cardiovascular (CV) system. Cardiovascular diseases (CVD) are worryingly one of the leading causes of all mortality and mobility worldwide. The UV-B filter octylmethoxycinnamate (also designated octinoxate, or ethylhexyl methoxycinnamate (CAS number: 5466-77-3)) is an EDC widely present in all personal care products. However, to date, there are no studies evaluating the OMC-induced effects on vasculature using animal models to improve human cardiovascular health. This work analysed the effects of OMC on rat aorta vasculature and explored the modes of action implicated in these effects. Our results indicated that OMC relaxes the rat aorta by endothelium-dependent mechanisms through the signaling pathways of cyclic nucleotides and by endothelium-independent mechanisms involving inhibition of L-Type voltage-operated Ca2+ channels (L-Type VOCC). Overall, OMC toxicity on rat aorta may produce hypotension via vasodilation due to excessive NO release and blockade of L-Type VOCC. Moreover, the OMC-induced endothelial dysfunction may also occur by promoting the endothelial release of endothelin-1. Therefore, our findings demonstrate that exposure to OMC alters the reactivity of the rat aorta and highlight that long-term OMC exposure may increase the risk of human CV diseases.

Discovery of isonucleotidic CDNs as potent STING agonists with immunomodulatory potential.

Stimulator of interferon genes (STING) is an adaptor protein of the cGAS-STING signaling pathway involved in the sensing of cytosolic DNA. It functions as a receptor for cyclic dinucleotides (CDNs) and, upon their binding, mediates cytokine expression and host immunity. Besides naturally occurring CDNs, various synthetic CDNs, such as ADU-S100, have been reported to effectively activate STING and are being evaluated in clinical trials for the treatment of cancer. Here, we describe the preparation of a unique new class of STING agonists: isonucleotidic cyclic dinucleotides and the synthesis of their prodrugs. The presented CDNs stimulate STING with comparable efficiency to ADU-S100, whereas their prodrugs demonstrate activity up to four orders of magnitude better due to the improved cellular uptake. The compounds are very potent inducers of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs). We also report the X-ray crystal structure of the lead inhibitor bound to the wild-type (WT) STING.

Roflumilast, a cyclic nucleotide phosphodiesterase 4 inhibitor, protects against cerebrovascular endothelial injury following cerebral ischemia/reperfusion by activating the Notch1/Hes1 pathway.

The loss of tight junction (TJ) and adherens junction (AJ) proteins leads to the damage of the blood-brain barrier (BBB) during cerebral ischemia. Inhibition of cyclic nucleotide phosphodiesterase 4 (PDE4) by roflumilast (Roflu) protects against ischemic stroke-induced neuronal damage. However, the effects of Roflu on vascular endothelial injury and BBB integrity remain unknown. Here, we investigated whether and how Roflu protects against cerebrovascular endothelial injury caused by cerebral ischemia/reperfusion. We demonstrated that PDE4B knocking-down increased the expression of TJ and AJ proteins in human brain microvascular endothelial cells (HBMECs) subjected to oxygen-glucose deprivation reperfusion (OGD/R). Inhibition of PDE4 by Roflu (1.0 µM) showed similar effects as PDE4B knocking-down. We then found that Roflu activated Notch1/Hairy and enhancer of split 1 (Hes1) signaling. Consistently, the effects of Roflu on TJ and AJ proteins were reversed by the γ-secretase inhibitor DAPT or Hes1 knocking-down. Furthermore, Roflu (1.0 mg/kg) improved neurobehavioral outcomes and ameliorated BBB disruption in rats following ischemic stroke. Roflu also increased the levels of TJ proteins and AJ proteins in vivo. Collectively, these data suggest that Roflu is a promising compound for the prevention of BBB damage. The protective effects of Roflu are mediated through activation of the Notch1/Hes1 pathway.

Effect of Riluzole on the Expression of HCN2 in Dorsal Root Ganglion Neurons of Diabetic Neuropathic Pain Rats.

Neuropathic pain since early diabetes swamps patients' lives, and diabetes mellitus has become an increasingly worldwide epidemic. No agent, so far, can terminate the ongoing diabetes. Therefore, strategies that delay the process and the further complications are preferred, such as diabetic neuropathic pain (DNP). Dysfunction of ion channels is generally accepted as the central mechanism of diabetic associated neuropathy, of which hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channel has been verified the involvement of neuropathic pain in dorsal root ganglion (DRG) neurons. Riluzole is a benzothiazole compound with neuroprotective properties on intervention to various ion channels, including hyperpolarization-activated voltage-dependent channels. To investigate the effect of riluzole within lumbar (L3-5) DRG neurons from DNP models, streptozocin (STZ, 70 mg/kg) injection was recruited subcutaneously followed by paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL), which both show significant reduction, whilst relieved by riluzole (4 mg/kg/d) administration, which was performed once daily for 7 consecutive days for 14 days. HCN2 expression was also decreased in line with alleviated behavioral tests. Our results indicate riluzole as the alleviator to STZ-induced DNP with involvement of downregulated HCN2 in lumbar DRG by continual systemic administration in rats.

STING agonist cGAMP enhances anti-tumor activity of CAR-NK cells against pancreatic cancer.

Activation of the stimulator of interferon gene (STING)-mediated innate immune response has been suggested as a promising therapeutic strategy for cancers. However, the effects of STING agonist on natural killer (NK) cell-mediated anti-tumor responses in pancreatic cancer remains unknown. Herein, we evaluated the effects of a classical STING agonist cyclic GMP-AMP (cGAMP) on NK cells in pancreatic cancer. We found that cGAMP could directly activate NK cells and enhance the sensitivity of pancreatic cancer cells to NK cell cytotoxicity, suggesting that cGAMP may become a potential adjuvant for NK cell therapy. In addition, combination of CAR-NK-92 cells targeting mesothelin and cGAMP displayed greater antitumor efficacy by inhibiting tumor growth and prolonging survival of the mouse model of pancreatic cancer. These results suggest that the combination of a STING agonist and NK cells may become a novel immunotherapy strategy for pancreatic cancer.

Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation in human platelets.

The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentrationdependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilatorstimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.

Impaired intestinal stem cell activity in ETEC infection: enterotoxins, cyclic nucleotides, and Wnt signaling.

Enterotoxigenic Escherichia coli (ETEC) in humans and animals colonizes the intestine and thereafter secrets heat-stable enterotoxin (ST) with or without heat-labile enterotoxin (LT), which triggers massive fluid and electrolyte secretion into the gut lumen. The crosstalk between the cyclic nucleotide-dependent protein kinase/cystic fibrosis transmembrane conductance regulator (cAMP or cGMP/CFTR) pathway involved in ETEC-induced diarrhea channels, and the canonical Wnt/ß-catenin signaling pathway leads to changes in intestinal stem cell (ISC) fates, which are strongly associated with developmental disorders caused by diarrhea. We review how alterations in enterotoxin-activated ion channel pathways and the canonical Wnt/ß-catenin signaling pathway can explain inhibited intestinal epithelial activity, characterize alterations in the crosstalk of cyclic nucleotides, and predict harmful effects on ISCs in targeted therapy. Besides, we discuss current deficits in the understanding of enterotoxin-intestinal epithelial cell activity relationships that should be considered when interpreting sequelae of diarrhea.