Novel Celecoxib Derivative, RF26, Blocks Colon Cancer Cell Growth by Inhibiting PDE5, Activating cGMP/PKG Signaling, and Suppressing ß-catenin-dependent Transcription.

BACKGROUND: Previous studies have reported that the cGMP-specific PDE5 isozyme is overexpressed in colon adenomas and adenocarcinomas and essential for colon cancer cell proliferation, while PDE5 selective inhibitors (e.g., sildenafil) have been reported to have cancer chemopreventive activity. AIM: This study aimed to determine the anticancer activity of a novel PDE5 inhibitor, RF26, using colorectal cancer (CRC) cells and the role of PDE5 in CRC tumor growth in vivo. OBJECTIVE: The objective of this study was to characterize the anticancer activity of a novel celecoxib derivative, RF26, in CRC cells previously reported to lack COX-2 inhibition but have potent PDE5 inhibitory activity. METHODS: Anticancer activity of RF26 was studied using human CRC cell lines. Its effects on intracellular cGMP levels, cGMP-dependent protein kinase (PKG) activity, ß-catenin levels, TCF/LEF transcriptional activity, cell cycle distribution, and apoptosis were measured. CRISPR/cas9 PDE5 knockout techniques were used to determine if PDE5 mediates the anticancer activity of RF26 and validate PDE5 as a cancer target. RESULTS: RF26 was appreciably more potent than celecoxib and sildenafil to suppress CRC cell growth and was effective at concentrations that increased intracellular cGMP levels and activated PKG signaling. RF26 suppressed ß-catenin levels and TCF/LEF transcriptional activity and induced G1 cell cycle arrest and apoptosis within the same concentration range. CRISPR/cas9 PDE5 knockout CRC cells displayed reduced sensitivity to RF26, proliferated slower than parental cells, and failed to establish tumors in mice. CONCLUSION: Further evaluation of RF26 for the prevention or treatment of cancer and studying the role of PDE5 in tumorigenesis are warranted.

Orthologs of Plasmodium ICM1 are dispensable for Ca2+ mobilization in Toxoplasma gondii.

Apicomplexan parasites mobilize ionic calcium (Ca2+) from intracellular stores to promote microneme secretion and facilitate motile processes including gliding motility, invasion, and egress. Recently, a multipass transmembrane protein, ICM1, was found to be important for calcium mobilization in Plasmodium falciparum and P. berghei. Comparative genomics and phylogenetics have revealed putative ICM orthologs in Toxoplasma gondii and other apicomplexans. T. gondii possesses two ICM-like proteins, which we have named TgICM1-L (TGGT1_305470) and TgICM2-L (TGGT1_309910). TgICM1-L and TgICM2-L localized to undefined puncta within the parasite cytosol. TgICM1-L and TgICM2-L are individually dispensable in tachyzoites, suggesting a potential compensatory relationship between the two proteins may exist. Surprisingly, mutants lacking both TgICM1-L and TgICM2-L are fully viable, exhibiting no obvious defects in growth, microneme secretion, invasion, or egress. Furthermore, loss of TgICM1-L, TgICM2-L, or both does not impair the parasite's ability to mobilize Ca2+. These findings suggest that additional proteins may participate in Ca2+ mobilization or import in Apicomplexa, reducing the dependence on ICM-like proteins in T. gondii. Collectively, these results highlight similar yet distinct mechanisms of Ca2+ mobilization between T. gondii and Plasmodium.IMPORTANCECa2+ signaling plays a crucial role in governing apicomplexan motility; yet, the mechanisms underlying Ca2+ mobilization from intracellular stores in these parasites remain unclear. In Plasmodium, the necessity of ICM1 for Ca2+ mobilization raises the question of whether this mechanism is conserved in other apicomplexans. Investigation into the orthologs of Plasmodium ICM1 in T. gondii revealed a differing requirement for ICM proteins between the two parasites. This study suggests that T. gondii employs ICM-independent mechanisms to regulate Ca2+ homeostasis and mobilization. Proteins involved in Ca2+ signaling in apicomplexans represent promising targets for therapeutic development.

Effect of Nitrosyl Iron Complex with 3,4-Dichlorothiophenolyls on the Level of Cyclic Nucleotide In Vitro.

The effect of a promising NO donor, a binuclear nitrosyl iron complex (NIC) with 3,4-dichlorothiophenolyls [Fe2(SC6H3Cl2)2(NO)4], on the adenylate cyclase and soluble guanylate cyclase enzymatic systems was studied. In in vitro experiments, this complex increased the concentration of important secondary messengers, such as cAMP and cGMP. An increase of their level by 2.4 and 4.5 times, respectively, was detected at NIC concentration of 0.1 mM. The ligand of the complex, 3,4-dichlorothiophenol, produced a less pronounced effect on adenylate cyclase. It was shown that the effect of this complex on the activity of soluble guanylate cyclase was comparable to the effect of anionic nitrosyl complex with thiosulfate ligands that exhibits vasodilating and cardioprotective properties.

Examining linaclotide for the treatment of chronic idiopathic constipation.

INTRODUCTION: Chronic idiopathic constipation (CIC) is characterized by infrequent bowel movements and hard stools lasting for at least three months or longer. This disease affects 8-12% of the US population and 10-17% of the world population. Treatment and management involve identifying the primary cause, changing dietary habits, and adequate physical activity. Linaclotide is a guanylate cyclase-agonist acting locally in the luminal surface of the intestinal enterocyte leading to a signal transduction cascade, activation of the cystic fibrosis transmembrane conductance regulator (CFTR), thus increasing secretion of chloride and bicarbonate into the intestinal lumen with eventual increased intestinal fluid and faster transit time. AREAS COVERED: We reviewed multiple studies and did a thorough literature review on CIC including its pathophysiology. Through this literature review, we were able to discuss and give the context and rationale for drug regimens indicated for CIC. EXPERT OPINION: The era we live in right now is akin to nutrient-rich and fertilized soil as knowledge and resources are abundant. The opportunities and potential are endless. Constipation being more extensively studied, our understanding of medications and diseases broadens, leading to novel medications being discovered. Linaclotide is a pioneer in this aspect and can pave the way for future generations.

Ca2+-dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Our studies reveal that SPNs manifest a heterosynaptic, nitric oxide (NO)-dependent form of long-term postsynaptic depression of glutamatergic SPN synapses (NO-LTD) that is preferentially engaged at quiescent synapses. Plasticity is gated by Ca2+ entry through CaV1.3 Ca2+ channels and phosphodiesterase 1 (PDE1) activation, which blunts intracellular cyclic guanosine monophosphate (cGMP) and NO signaling. Both experimental and simulation studies suggest that this Ca2+-dependent regulation of PDE1 activity allows for local regulation of dendritic cGMP signaling. In a mouse model of Parkinson disease (PD), NO-LTD is absent because of impaired interneuronal NO release; re-balancing intrastriatal neuromodulatory signaling restores NO release and NO-LTD. Taken together, these studies provide important insights into the mechanisms governing NO-LTD in SPNs and its role in psychomotor disorders such as PD.

Considerations for the development of iPSC-derived cell therapies: a review of key challenges by the JSRM-ISCT iPSC Committee.

Since their first production in 2007, human induced pluripotent stem cells (iPSCs) have provided a novel platform for the development of various cell therapies targeting a spectrum of diseases, ranging from rare genetic eye disorders to cancer treatment. However, several challenges must be tackled for iPSC-based cell therapy to enter the market and achieve broader global adoption. This white paper, authored by the Japanese Society for Regenerative Medicine (JSRM) - International Society for Cell Therapy (ISCT) iPSC Committee delves into the hurdles encountered in the pursuit of safe and economically viable iPSC-based therapies, particularly from the standpoint of the cell therapy industry. It discusses differences in global guidelines and regulatory frameworks, outlines a series of quality control tests required to ensure the safety of the cell therapy, and provides details and important considerations around cost of goods (COGs), including the impact of automated advanced manufacturing.

Isopsoralen Improves Glucocorticoid-induced Osteoporosis by Regulating Purine Metabolism and Promoting cGMP/PKG Pathway-mediated Osteoblast Differentiation.

BACKGROUND: The effects of Isopsoralen (ISO) in promoting osteoblast differentiation and inhibiting osteoclast formation are well-established, but the mechanism underlying ISO's improvement of Glucocorticoid- Induced Osteoporosis (GIOP) by regulating metabolism remains unclear. METHODS: This study aims to elucidate the mechanism of ISO treatment for GIOP through non-targeted metabolomics based on ISO's efficacy in GIOP. Initially, we established a GIOP female mouse model and assessed ISO's therapeutic effects using micro-CT detection, biomechanical testing, serum calcium (Ca), and phosphorus (P) level detection, along with histological analyses using hematoxylin and eosin (HE), Masson, and tartrate-resistant acidic phosphatase (TRAP) staining. Subsequently, non-targeted metabolomics was employed to investigate ISO's impact on serum metabolites in GIOP mice. RT-qPCR and Western blot analyses were conducted to measure the levels of enzymes associated with these metabolites. Building on the metabolomic results, we explored the effects of ISO on the cyclic Guanosine Monophosphate (cGMP)/Protein Kinase G (PKG) pathway and its role in mediating osteoblast differentiation. RESULTS: Our findings demonstrate that ISO intervention effectively enhances the bone microarchitecture and strength of GIOP mice. It mitigates pathological damage, such as structural damage in bone trabeculae, reduced collagen fibers, and increased osteoclasts, while improving serum Ca and P levels in GIOP mice. Non-- targeted metabolomics revealed purine metabolism as a common pathway between the Control and GIOP groups, as well as between the ISO high-dose (ISOH) group and the GIOP group. ISO intervention upregulated inosine and adenosine levels, downregulated guanosine monophosphate levels, increased Adenosine Deaminase (ADA) expression, and decreased cGMP-specific 3',5'-cyclic phosphodiesterase (PDE5) expression. Additionally, ISO intervention elevated serum cGMP levels, upregulated PKGI and PKGII expression in bone tissues, as well as the expression of Runt-related transcription factor 2 (Runx2) and Osterix, and increased serum Alkaline Phosphatase (ALP) activity. CONCLUSION: In summary, ISO was able to enhance the bone microstructure and bone strength of GIOP mice and improve their Ca, P, and ALP levels, which may be related to ISO's regulation of purine metabolism and promotion of osteoblast differentiation mediated by the cGMP/PKG pathway. This suggests that ISO is a potential drug for treating GIOP. However, further research is still needed to explore the specific targets and clinical applications of ISO.

Analysis of cGMP Signaling in Dictyostelium.

Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.

Inflammation-Related Gene ADH1A Regulates the Polarization of Macrophage M1 and Influences the Malignant Progression of Gastric Cancer.

Background: Gastric cancer (GC) is a malignant tumor originating from the gastric mucosa epithelium, and there is a low survival rate of GC patients after treatment, with a poor prognostic outcome. The inflammatory response within the tumor microenvironment plays an important role in GC progression. Methods: We downloaded GC-related datasets and inflammation-related genes from GEO, TCGA and MSigDB databases, performed differential analysis, protein-protein interaction analysis, immunoinfiltration analysis and Lasso analysis to screen inflammation-related hub genes affecting GC progression, and carried out qRT-PCR for validation. In order to explore the role of ADH1A, we constructed overexpressed plasmids, treated GC cells with cGMP/PKG pathway agonist 8-Br-cGMP, and tested cell functions with CCK8, EdU, Transwell, scratch assay and other experiments. On this basis, GC cells were co-cultured with monocyte THP-1 to explore the effect of ADH1A on the polarization of macrophages. Results: ADH1A was significantly decreased in GC cells, and its expression trend was consistent with the results of bioinformatics analysis. Therefore, we chose ADH1A for subsequent functional validation. Overexpression of ADH1A in GC cells revealed ADH1A's role in inhibiting the activity, proliferation, migration and invasion of GC cells, promoting apoptosis and secretion of IL-6, IFN-γ, CCL5 and CSF2, and facilitating the transformation of macrophages to a pro-inflammatory M1 phenotype. ssGSEA results demonstrated the potential involvement of ADH1A in the cGMP/PKG signaling pathway, and significant changes in the expression of proteins related to the cGMP/PKG signaling pathway. The use of the cGMP/PKG signaling pathway agonist 8-Br-cGMP in ADH1A-overexpressing GC cells substantiated ADH1A's capacity to inhibit the cGMP/PKG signaling pathway, thereby suppressing the malignant progression of GC and promoting the transformation of macrophages to a pro-inflammatory M1 phenotype. Conclusion: ADH1A is able to influence the malignant progression of GC and the transformation of macrophages to the pro-inflammatory M1 phenotype through the cGMP/PKG signaling pathway.

Placental Protein 13: Vasomodulatory Effects on Human Uterine Arteries and Potential Implications for Preeclampsia.

Placental protein 13 (PP13) exhibits a plasma concentration that increases gradually during normal gestation, a process that is disrupted in preeclampsia, which is characterized by elevated vascular resistance, reduced utero-placental blood flow, and intrauterine growth restriction. This study investigated PP13's role in vascular tone regulation and its molecular mechanisms. Uterine and subcutaneous arteries, isolated from both pregnant and non-pregnant women, were precontracted with the thromboxane analogue U46619 and exposed to PP13 using pressurized myography. The molecular mechanisms were further investigated, using specific inhibitors for nitric oxide synthase (L-NAME+LNNA at 10-4 M) and guanylate cyclase (ODQ at 10-5 M). The results showed that PP13 induced vasodilation in uterine arteries, but not in subcutaneous arteries. Additionally, PP13 counteracted U46619-induced vasoconstriction, which is particularly pronounced in pregnancy. Further investigation revealed that PP13's mechanism of action is dependent on the activation of the nitric oxide-cGMP pathway. This study provides novel insights into the vasomodulatory effects of PP13 on human uterine arteries, underscoring its potential role in regulating utero-placental blood flow. These findings suggest that PP13 may be a promising candidate for improving utero-placental blood flow in conditions such as preeclampsia. Further research and clinical studies are warranted to validate PP13's efficacy and safety as a therapeutic agent for managing preeclampsia.

Vasorelaxant and Hypotensive Effects of Galla chinensis in Rats.

Previous studies have revealed the medicinal and therapeutic effects of Galla chinensis. However, no studies have focused on the antihypertensive effects of G. chinensis. Therefore, we aimed to determine the vasorelaxant and hypotensive effects of G. chinensis 50% ethanolic extract (GCE). To evaluate the vascular relaxing effect of GCE, experiments were conducted using aortic segments dissected from Sprague Dawley rats. GCE showed a vasorelaxant effect via the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway, inhibiting Ca2+ channels, and activating K+ channels. The hypotensive effects of GCE were evaluated in spontaneously hypertensive rats (SHRs). The SHRs were randomly divided into a control group and orally administered GCE group (100 or 300 mg/kg). The systolic and diastolic blood pressure decreased significantly by -19.47 ± 4.58% and -31.14 ± 7.66% in the GCE 100 mg/kg group, and -21.64 ± 2.40% and -31.91 ± 5.75% in the GCE 300 mg/kg group at 4 h after administration. Considering its vasorelaxant and hypotensive effects, our results indicate that GCE may be a valuable solution for the control of hypertension. However, further studies on the long-term administration and toxicity of GCE are required.

Inonotus obliquus (Chaga) against HFD/STZ-induced glucolipid metabolism disorders and abnormal renal functions by regulating NOS-cGMP-PDE5 signaling pathway.

Our prior investigations have established that Inonotus obliquus (Chaga) possesses hypoglycemic effects. Persistent hyperglycemia is known to precipitate renal function abnormalities. The functionality of the kidneys is intricately linked to the levels of cyclic guanosine-3',5'-monophosphate (cGMP), which are influenced by the activities of nitric oxide synthase (NOS) and phosphodiesterase (PDE). Enhanced cGMP levels can be achieved either through the upregulation of NOS activity or the downregulation of PDE activity. The objective of the current study is to elucidate the effects of Chaga on disorders of glucolipid metabolism and renal abnormalities in rats with type 2 diabetes mellitus (T2DM), while concurrently examining the NOS-cGMP-PDE5 signaling pathway. A model of T2DM was developed in rats using a high-fat diet (HFD) combined with streptozotocin (STZ) administration, followed by treatment with Chaga extracts at doses of 50 and 100 mg·kg-1 for eight weeks. The findings revealed that Chaga not only mitigated metabolic dysfunctions, evidenced by improvements in fasting blood glucose, total cholesterol, triglycerides, and insulin resistance, but also ameliorated renal function markers, including serum creatinine, urine creatinine (UCr), blood urea nitrogen, 24-h urinary protein, and estimated creatinine clearance. Additionally, enhancements in glomerular volume, GBM thickness, podocyte foot process width (FPW), and the mRNA and protein expressions of podocyte markers, such as nephrin and wilms tumor-1, were observed. Chaga was found to elevate cGMP levels in both serum and kidney tissues by increasing mRNA and protein expressions of renal endothelial NOS and neural NOS, while simultaneously reducing the expressions of renal inducible NOS and PDE5. In summary, Chaga counteracts HFD/STZ-induced glucolipid metabolism and renal function disturbances by modulating the NOS-cGMP-PDE5 signaling pathway. This research supports the potential application of Chaga in the clinical prevention and treatment of T2DM and diabetic nephropathy (DN), with cGMP serving as a potential therapeutic target.

Unresolved mystery of cyclic nucleotide second messengers, periplasmic acid phosphatases and bacterial natural competence.

We recently characterized the competitive inhibition of cyclic AMP (cAMP) on three periplasmic acid phosphatases, AphAHi, NadNHi, and eP4 (HelHi), in Haemophilus influenzae Rd KW20. This inhibitory effect is vital for orchestrating the nutritional growth and competence development in KW20. Initially discovered in Escherichia coli, the function of AphA remains however obscure. This study investigates the regulation of E. coli aphA expression under nutrient starvation conditions. Using transcriptional reporters with truncated aphA promoter sequences, we found that starvations of carbon and phosphate, but not amino acid, stimulated aphA expression through distinct promoter regions. Deletions of crp or cyaA abolished aphA expression, confirming their crucial roles. Conversely, CytR deletion increased aphA expression, suggesting CytR's role as a repressor of aphA expression. Additionally, we extended the study of three other second messengers, i.e., cyclic GMP, cyclic UMP, and cyclic CMP, each sharing structural similarities with cAMP. Notably, cGMP competitively inhibits AphAHi's acid phosphatase activity akin to cAMP. In contrast, both cUMP and cCMP stimulate AphAHi's phosphatase activity in a concentration dependent manner. Collectively, these data imply a complicated connection between nucleotide metabolism, AphA, cyclic purine and pyrimidine nucleotides in bacterial nutrient uptake and natural competence.

The multidrug resistance protein 4 is expressed and functionally active in isolated bladder from pig.

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-precontracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.NEW & NOTEWORTHY This study investigates the impact of pharmacological inhibition of MRP4 and MRP5 transporters on cyclic nucleotide signaling in isolated pig bladders. MK571 administration led to modest relaxation, with enhanced effects observed in the presence of phosphodiesterase inhibitors. However, substances elevating cAMP levels remained unaffected. MK571 selectively reduced extracellular cGMP levels. These findings shed light on the role of MRP4 transporters in the porcine bladder, opening avenues for further research into bladder disorders.

Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.

The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The present study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs), indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type, whereas the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (l-NNA) abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by l-NNA but not MRS2500, suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1R) and small-conductance Ca2+-activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS, with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC, and PDGFRα+ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).

Discovery of small molecule guanylyl cyclase B receptor positive allosteric modulators.

Myocardial fibrosis is a pathological hallmark of cardiovascular disease (CVD), and excessive fibrosis can lead to new-onset heart failure and increased mortality. Currently, pharmacological therapies for myocardial fibrosis are limited, highlighting the need for novel therapeutic approaches. The particulate guanylyl cyclase B (GC-B) receptor possesses beneficial antifibrotic actions through the binding of its natural ligand C-type natriuretic peptide (CNP) and the generation of the intracellular second messenger, cyclic guanosine 3',5'-monophosphate (cGMP). These actions include the suppression of fibroblast proliferation and reduction in collagen synthesis. With its abundant expression on fibroblasts, the GC-B receptor has emerged as a key molecular target for innovative CVD therapeutics. However, small molecules that can bind and potentiate the GC-B/cGMP pathway have yet to be discovered. From a cell-based high-throughput screening initiative of the NIH Molecular Libraries Small Molecule Repository and hit-to-lead evolution based on a series of structure-activity relationships, we report the successful discovery of MCUF-42, a GC-B-targeted small molecule that acts as a positive allosteric modulator (PAM). Studies herein support MCUF-42's ability to enhance the binding affinity between GC-B and CNP. Moreover, MCUF-42 potentiated cGMP levels induced by CNP in human cardiac fibroblasts (HCFs) and notably also enhanced the inhibitory effect of CNP on HCF proliferation. Together, our findings highlight that MCUF-42 is a small molecule that can modulate the GC-B/cGMP signaling pathway, potentially enhancing the antifibrotic actions of CNP. Thus, these data underscore the continued development of GC-B small molecule PAMs as a novel therapeutic strategy for targeting cardiac fibrosis and CVD.

Regulatory Role of Fatty Acid Metabolism on Glucose-Induced Changes in Insulin and Glucagon Secretion by Pancreatic Islet Cells.

A detailed study of palmitate metabolism in pancreatic islets subject to different experimental conditions, like varying concentrations of glucose, as well as fed or starved conditions, has allowed us to explore the interaction between the two main plasma nutrients and its consequences on hormone secretion. Palmitate potentiates glucose-induced insulin secretion in a concentration-dependent manner, in a physiological range of both palmitate (0-2 mM) and glucose (6-20 mM) concentrations; at glucose concentrations lower than 6 mM, no metabolic interaction with palmitate was apparent. Starvation (48 h) increased islet palmitate oxidation two-fold, and the effect was resistant to its inhibition by glucose (6-20 mM). Consequently, labelled palmitate and glucose incorporation into complex lipids were strongly suppressed, as well as glucose-induced insulin secretion and its potentiation by palmitate. 2-bromostearate, a palmitate oxidation inhibitor, fully recovered the synthesis of complex lipids and insulin secretion. We concluded that palmitate potentiation of the insulin response to glucose is not attributable to its catabolic mitochondrial oxidation but to its anabolism to complex lipids: islet lipid biosynthesis is dependent on the uptake of plasma fatty acids and the supply of α-glycerol phosphate from glycolysis. Islet secretion of glucagon and somatostatin showed a similar dependence on palmitate anabolism as insulin. The possible mechanisms implicated in the metabolic coupling between glucose and palmitate were commented on. Moreover, possible mechanisms responsible for islet gluco- or lipotoxicity after a long-term stimulation of insulin secretion were also discussed. Our own data on the simultaneous stimulation of insulin, glucagon, and somatostatin by glucose, as well as their modification by 2-bromostearate in perifused rat islets, give support to the conclusion that increased FFA anabolism, rather than its mitochondrial oxidation, results in a potentiation of their stimulated release. Starvation, besides suppressing glucose stimulation of insulin secretion, also blocks the inhibitory effect of glucose on glucagon secretion: this suggests that glucagon inhibition might be an indirect or direct effect of insulin, but not of glucose. In summary, there seems to exist three mechanisms of glucagon secretion stimulation: 1. glucagon stimulation through the same secretion coupling mechanism as insulin, but in a different range of glucose concentrations (0 to 5 mM). 2. Direct or indirect inhibition by secreted insulin in response to glucose (5-20 mM). 3. Stimulation by increased FFA anabolism in glucose intolerance or diabetes in the context of hyperlipidemia, hyperglycemia, and hypo-insulinemia. These conclusions were discussed and compared with previous published data in the literature. Specially, we discussed the mechanism for inhibition of glucagon release by glucose, which was apparently contradictory with the secretion coupling mechanism of its stimulation.

The activation of Chlamydomonas reinhardtii alpha amylase 2 by glutamine requires its N-terminal aspartate kinase-chorismate mutase-tyrA (ACT) domain.

The coordination of assimilation pathways for all the elements that make up cellular components is a vital task for every organism. Integrating the assimilation and use of carbon (C) and nitrogen (N) is of particular importance because of the high cellular abundance of these elements. Starch is one of the most important storage polymers of photosynthetic organisms, and a complex regulatory network ensures that biosynthesis and degradation of starch are coordinated with photosynthetic activity and growth. Here, we analyzed three starch metabolism enzymes of Chlamydomonas reinhardtii that we captured by a cyclic guanosine monophosphate (cGMP) affinity chromatography approach, namely, soluble starch synthase STA3, starch-branching enzyme SBE1, and α-amylase AMA2. While none of the recombinant enzymes was directly affected by the presence of cGMP or other nucleotides, suggesting an indirect binding to cGMP, AMA2 activity was stimulated in the presence of L-glutamine (Gln). This activating effect required the enzyme's N-terminal aspartate kinase-chorismate mutase-tyrA domain. Gln is the first N assimilation product and not only a central compound for the biosynthesis of N-containing molecules but also a recognized signaling molecule for the N status. Our observation suggests that AMA2 might be a means to coordinate N and C metabolism at the enzymatic level, increasing the liberation of C skeletons from starch when high Gln levels signal an abundance of assimilated N.

Haemodynamic, hormonal and renal actions of osteocrin in normal sheep.

Osteocrin (OSTN) is an endogenous protein sharing structural similarities with the natriuretic peptides [NPs; atrial (ANP), B-type (BNP) and C-type (CNP) NP], which are hormones known for their crucial role in maintaining pressure/volume homeostasis. Osteocrin competes with the NPs for binding to the receptor involved in their clearance (NPR-C). In the present study, having identified, for the first time, the major circulating form of OSTN in human and ovine plasma, we examined the integrated haemodynamic, endocrine and renal effects of vehicle-controlled incremental infusions of ovine proOSTN (83-133) and its metabolism in eight conscious normal sheep. Incremental i.v. doses of OSTN produced stepwise increases in circulating concentrations of the peptide, and its metabolic clearance rate was inversely proportional to the dose. Osteocrin increased plasma levels of ANP, BNP and CNP in a dose-dependent manner, together with concentrations of their intracellular second messenger, cGMP. Increases in plasma cGMP were associated with progressive reductions in arterial pressure and central venous pressure. Plasma cAMP, renin and aldosterone were unchanged. Despite significant increases in urinary cGMP levels, OSTN administration was not associated with natriuresis or diuresis in normal sheep. These results support OSTN as an endogenous ligand for NPR-C in regulating plasma concentrations of NPs and associated cGMP-mediated bioactivity. Collectively, our findings support a role for OSTN in maintaining cardiovascular homeostasis.