cGMP Levels Research Articles

Increased Responsiveness of Stored Platelets after Short-Term Refrigeration

Introduction: Refrigeration of platelets is considered to provide advantages in therapy of acute hemorrhage due to increased platelet responsiveness. The alleviation of inhibitory signaling caused by cold temperature (CT) has been identified as an important mechanism contributing to enhanced platelet reactivity, detectable in freshly prepared platelets within 1 h of cold storage. The aim of this study was to confirm the effects of short-term refrigeration in platelets from apheresis-derived platelet concentrates (APC). Methods: APC were stored under standardized conditions for 1 day or for 2 days at room temperature and then refrigerated for 1 h, followed by sampling of platelets for analysis. Platelet reactivity was measured by aggregation studies using threshold concentrations of different agonists and by detection of fibrinogen binding using flow cytometry. The exploration of inhibitory signaling comprised the detection of VASP phosphorylation using flow cytometry or Western blot and the measurement of cyclic nucleotide levels. Results: Aggregation responses induced with ADP, collagen, or thrombin receptor-activating peptide-6 (TRAP-6) were increased in APC after cold storage for 1 h, associated with elevated TRAP-6-induced fibrinogen binding. VASP phosphorylation levels were decreased after cold exposition, detectable in 1-day- and 2-day-stored APC with flow cytometry, and in 2-day-stored APC with Western blot technique. Induced cGMP levels were lower after storage at CT in APC on day 1 and on day 2, whereas cAMP levels were reduced on 2-day-stored APC. Conclusion: Short-term refrigeration for 1 h is sufficient to induce an attenuation of inhibitory signaling, accompanied with increased aggregation responses in APC stored for up to 2 days. The “on demand” refrigeration of PC may be a reasonable approach for the preparation of platelets with enhanced responsiveness to treat patients with hemorrhage more effectively, which should be further addressed in consecutive studies.

Vasoactive mediators of hypertension in obesity.

Obesity is associated with hypertension. However, the mechanisms involved are not fully understood. Therefore, we investigated the relationship between obesity and vasoactive mediators. In this cross-sectional study, blood pressure (BP) and vasoactive mediators of hypertension are compared among 135 adults in the nonobese, obese, and morbidly obese body mass index (BMI) ranges (BMI ≤27, 30-40, and >40 kg/m2, respectively). Angiotensinogen, angiotensin II, renin, aldosterone, endothelin-1 (ET-1), neprilysin, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cyclic guanosine monophosphate (cGMP), and cyclic adenosine monophosphate (cAMP) levels were measured and their relationship to BP, BMI, race, and gender were investigated. Systolic and diastolic BP (SBP and DSP) were significantly higher in subjects with obesity and morbid obesity compared with nonobese. Angiotensin II, ET-1, and neprilysin were significantly higher in subjects with morbid obesity while BNP was lower. Levels of angiotensinogen, renin, aldosterone, ANP, cGMP, and cAMP did not differ between the groups. BMI was positively related to SBP, DBP, angiotensin II, ET-1, and neprilysin, and inversely related to cGMP and BNP. Age, male gender, and African-American race were associated with higher SBP. SBP was positively related to angiotensin II and ET-1 and inversely related to aldosterone, renin, and cGMP. On multivariate analyses, age, BMI, gender, and race were the main determinants of SBP, and excluding these variables, angiotensin II, aldosterone, renin, and ET-1 accounted for 21.1% ability to predict SBP. Obesity, especially morbid obesity, is associated with higher BP, higher angiotensin II and ET-1 (vasoconstrictors), and lower levels BNP and cGMP (vasodilators). SBP variability can be partly explained by angiotensin II, aldosterone, renin, and ET-1.NEW & NOTEWORTHY Our data show that obesity, especially morbid obesity, is associated with higher blood pressure levels and increases angiotensin II and endotherlin-1 (ET-1) (vasoconstrictors) and lower levels BNP and cGMP (vasodilators) and that systolic blood pressure variability can be partly explained by levels of angiotensin II, aldosterone, renin, and ET-1. The effect of these mediators on blood pressure is in addition to the effects of other known factors related to age, male gender, and AA race.

Volatile anesthetics inhibit presynaptic cGMP signaling to depress presynaptic excitability in rat hippocampal neurons

Volatile anesthetics alter presynaptic function through effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including immobility, unconsciousness and amnesia. Nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated volatile anesthetic actions NO/cGMP signaling in relation to presynaptic function in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors and sequestering probes of cGMP levels. Using the fluorescent cGMP biosensor cGull, we found that electrical stimulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited 33.2% by isoflurane (0.51 mM) and 26.4% by sevoflurane (0.57 mM) (p < 0.0001) compared to control stimulation without anesthetic. Stimulation-evoked cGMP transients were blocked by the nonselective inhibitor of nitric oxide synthase N-ω-nitro-l-arginine, but not by the selective neuronal nitric oxide synthase inhibitor N5-(1-imino-3-butenyl)-l-ornithine. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and of synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, was attenuated in neurons expressing the cGMP scavenger protein sponge (inhibition of exocytosis reduced by 54% for isoflurane and by 53% for sevoflurane). The anesthetic-induced reduction in presynaptic excitability was partially occluded by inhibition of HCN channels, a cGMP-modulated excitatory ion channel that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify novel mechanisms by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway in hippocampal neurons.

Extract of Pfaffia glomerata Ameliorates Paroxetine-Induced Sexual Dysfunction in Male Mice and the Characterization of Its Phytoconstituents by UPLC-MS.

Pfaffia glomerata extract (PGE) has a variety of biological activities. However, its ameliorative effect on and exact working mechanism in male sexual dysfunction are still poorly understood. This study aims to evaluate the ameliorative effect of PGE on paroxetine (PRX)-induced sexual dysfunction in male mice and uses molecular docking technology to investigate its underlying mechanism. In this work, PRX-induced sexual dysfunction was caused and PGE was gavaged in mice for 28 days. The results show that PGE significantly improved the sexual performance of mice and reduced the damage to testicular tissues. Further studies showed that PGE restored serum sex hormones to normal levels and increased nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels as well as nitric oxide synthase (NOS) activity in penile tissues, while also decreasing phosphodiesterase-5 (PDE-5) activity, thereby maintaining normal penile erection in mice. In addition, PGE improved the activities of enzymes (LDH, ACP, and ALP) related to energy metabolism in the testis and significantly increased sperm count and viability in mice. Furthermore, the molecular docking results show that all eight compounds in PGE could form a stable complex with PDE-5 and inhibit the activity of PDE-5. In conclusion, PGE had an ameliorative effect on PRX-induced sexual dysfunction, suggesting that PGE has a potential protective effect on male sexual health.

Cyclic guanosine monophosphate improves salt tolerance in Solanum lycopersicum

The cyclic nucleotide cyclic guanosine monophosphate (cGMP) is a powerful cell signaling molecule involved in biotic and abiotic stress perception and signal transduction. In the model plant Arabidopsis thaliana, salt and osmotic stress rapidly induce increase in cGMP which plays role by modulating the activity of monovalent cation transporters, possibly by direct binding to these proteins and by altering the expression of many abiotic stress responsive genes. In a recent study, a membrane permeable analogue of cGMP (8-bromo-cGMP) was found to have a promotive effect on soluble sugar, flavonoids and lignin content, and membrane integrity in Solanum lycopersicum seedlings under salt stress. However, it remains to be elucidated how salt stress affects the endogenous cGMP level in S. lycopersicum and if Br-cGMP-induced improvement in salt tolerance in S. lycopersicum involves altered cation fluxes. The current study was conducted to answer these questions. A rapid increase (within 30 s) in endogenous cGMP level was determined in S. lycopersicum roots after treatment with 100 mM NaCl. Addition of membrane permeable Br-cGMP in growth medium remarkably ameliorated the inhibitory effects of NaCl on seedlings’ growth parameters, chlorophyll content and net photosynthesis rate. In salt stressed plants, Br-cGMP significantly decreased Na+ content by reducing its influx and increasing efflux while it improved plants K+ content by reducing its efflux and enhancing influx. Furthermore, supplementation with Br-cGMP improved plant’s proline content and total antioxidant capacity, resulting in markedly decreased electrolyte leakage under salt stress. Br-cGMP increased the expression of Na+/H+ antiporter genes in roots and shoots of S. lycopersicum growing under salt stress, potentially enhancing plant’s ability to sequester Na+ into the vacuole. The findings of this study provide insights into the mechanism of cGMP-induced salt stress tolerance in S. lycopersicum.

Corin deficiency impairs cardiac function in mouse models of heart failure.

Corin is a protease in the natriuretic peptide system. Deleterious CORIN variants are associated with hypertension and heart disease. It remains unclear if and to what extent corin deficiency may contribute to heart failure (HF). Corin knockout (KO) mice were used as a model. Cardiac function was assessed by echocardiography and tissue analysis in Corin KO mice at different ages or subjected to transverse aortic constriction (TAC), which increased pressure overload. Heart and lung tissues were analyzed for cardiac hypertrophy and lung edema using wheat germ agglutinin, Sirius red, Masson's trichrome, and Prussian blue staining. Recombinant corin was tested for its effect on cardiac function in the TAC-operated Corin KO mice. Selected gene expression in the heart was examined by RT-PCR. ELISA was used to analyze factors in plasma. Corin KO mice had progressive cardiac dysfunction with cardiac hypertrophy and fibrosis after 9 months of age, likely due to chronic hypertension. When Corin KO mice were subjected to TAC at 10-12 weeks of age, cardiac function decreased more rapidly than in similarly treated wild-type mice. When the TAC-operated Corin KO mice were treated with recombinant corin protein, cardiac dysfunction, hypertrophy, and fibrosis were ameliorated. The corin treatment also decreased the gene expression associated with cardiac hypertrophy and fibrosis, increased plasma cGMP levels, lowered plasma levels of N-terminal pro-atrial natriuretic peptide, angiotensin II, and aldosterone, and lessened lung edema in the Corin KO mice subjected to TAC. Corin deficiency impairs cardiac function and exacerbates HF development in mice. Corin protein may be used to reduce cardiac hypertrophy and fibrosis, suppress the renin-angiotensin-aldosterone system, and improve cardiac function in HF.

Effects of transplantation of umbilical cord blood mononuclear cells into the scrotum on sexual function in elderly mice.

Aim: This study investigated the effect of allografting umbilical cord blood mononuclear cells (UCBMCs) into the scrotum on sexual function in male elderly mice. Methods: UCBMCs were injected once into the scrotal sheath cavity of elderly mice. Results: The transplanted UCBMCs survived in the scrotal sheath cavity for 1month. The micehad significantly increased blood testosterone concentrations, cyclic guanosine monophosphate (cGMP) levels and total nitric oxide synthase (T-NOS) activity in the corpus cavernosum and an increase inthe number of mouse matings within 30min (all p=0.000). Conclusion: Scrotum-implanted UCBMCs improve the sexual function of male elderly mice through testosterone production and the NOS/cGMP pathway, which may provide an innovative transplantation approach for thetreatment of erectile dysfunction.

Buxu Tongyu Granule Alleviates Myocardial Ischemia by Activating Vascular Smooth Muscle Cell Soluble Guanylate Cyclase to Inhibit Abnormal Vasomotion

Myocardial ischemia is a serious threat to human health, and vascular dysfunction is its main cause. Buxu Tongyu (BXTY) Granule is an effective traditional Chinese medicine (TCM) for treating myocardial ischemia. However, the underlying mechanism of BXTY is still unclear. In this study, we demonstrate that BXTY ameliorates myocardial ischemia by activating the soluble guanylate cyclase (sGC)–3′,5′-cyclic guanosine monophosphate (cGMP)–protein kinase G (PKG) signaling pathway in vascular smooth muscle cells (VSMCs) to dilate the arteries. BXTY was given by gavage for ten consecutive days before establishing an animal model of acute myocardial ischemia in mice via the intraperitoneal injection of pituitrin. The results showed that BXTY alleviated the symptoms of myocardial ischemia induced by pituitrin in mice, including electrocardiogram abnormalities and changes in plasma enzymes. In addition, BXTY dilated pre-constricted blood vessels and inhibited the vasoconstriction of the superior mesenteric artery in a dose-dependent but endothelial-independent manner. These effects were eliminated by pre-incubating vascular rings with the sGC inhibitors NS 2028 or ODQ, or with the PKG inhibitor KT 5823. Moreover, BXTY increased the protein expression of sGC-β1 and the intracellular second messenger cGMP level in mouse aortic vascular smooth muscle cells (MOVAs). NS 2028 or ODQ reversed these effects of BXTY. The expression level of the cGMP downstream effector protein PKG-1 increased after treating MOVAs with BXTY. NS 2028, ODQ, or KT 5823 also reversed this effect of BXTY. In conclusion, BXTY can improve the symptoms of acute myocardial ischemia in mice, and activating the sGC–cGMP–PKG pathway in VSMCs to induce vasodilation is its key pharmacodynamic mechanism.

Abstract P3128: Synapse-associated Protein 97 Is Necessary For Enhancing Cardiac Contractility Via Beta1 Adrenoceptor-neuronal Nitric Oxide Synthase Pathway And Guanosine3',5'-cyclic Monophosphate Signaling

Objective: Synapse-associated protein 97 (SAP97) is a scaffold protein widely expressed in the brain, heart, and kidney. SAP97 has been suggested function in the regulation of cardiac contractility. SAP97 directly binds to cardiac beta1 adrenceptor. Our recent studies have shown that increased cAMP and cGMP levels are involved in enhancing excitation-contraction (E-C) coupling after sympathetic nervous system activation. We aim to study the role of SAP97 in beta1 adrenoceptor-induced increases in cyclic nucleotides and cardiac E-C coupling. Results and Methods: Cardiac function in mice with cardiomyocyte-specific deletion of the SAP97 (SAP97-cKO) was comparable to that in flox/flox control littermates. Ligand-induced cGMP signal in freshly isolated adult ventricular myocytes (AVMs) using FRET biosensor Gi500. The cGMP responsiveness caused by β1AR agonist as dobutamine was reduced in SAP97-cKO versus f/f cells. No significant difference between the groups using nonselective beta-adrenergic stimulation isoproterenol (ISO). In comparison, the dobutamine-induced cAMP levels were not reduced in SAP97-cKO when compared to f/f controls. The dobutamine-induced ejection fraction in hearts and sarcomere shortening in AVMs were significantly attenuated in SAP97-cKO hearts and AVMs when compared to f/f controls, respectively. On the other hand, no difference was observed between f/f and cKO AVMs and hearts after stimulation by ISO. To further examine the LTCC activity, whole-cell currents (ICa) were recorded from freshly isolated AVMs. The results showed that the LTCC current was attenuated in SAP97-cKO AVMs than cKO controls when exposed to dobutamine. These results show SAP97 is necessary for increasing intracellular cGMP level, LTCC current, and cardiac E-C coupling and contractility after β1AR stimulation. Conclusion: SAP97 plays a crucial role in β 1 AR signaling, which is necessary for triggering E-C coupling through cAMP and cGMP pathways.

Traditional Chinese Medicine formula Wubi Shanyao Pills protects against reproductive aging by activating SIRT1/3 to reduce apoptosis

Ethnopharmacological relevanceThe study of male reproductive aging and its associated concerns holds significant importance within the realm of health issues affecting the elderly population. Wubi Shanyao Pills (WSP), a traditional Chinese patent medicine originating from the Tang Dynasty, has been recognized for its ability to enhance male sexual functions while also tonifying the kidney and spleen. Nevertheless, the precise effects and underlying mechanisms through which WSP ameliorates the decline in reproductive function among aging men remain uncertain. Aim of the studyThis study elucidated the distinctive impacts of WSP on ameliorating the decline in reproductive function caused by natural aging, as well as its underlying mechanisms. Materials and methodsInitially, male mice at the age of 15 months were administered WSP orally at doses of 0.375, 0.75, and 1.50 g/kg per day for a duration of 8 consecutive weeks. The impact of WSP on age-related manifestations in naturally aging mice was assessed based on their behavioral performance. The renal function of the mice was evaluated by measuring serum biochemical indicators, including Creatinine (CR), Uric acid (UA), and Blood urea nitrogen (BUN). Additionally, Superoxide dismutase (SOD) and Malonaldehyde (MDA) levels in renal tissue were determined using applied chemistry methods. Then assessed the levels of Nitric oxide (NO), Total nitric oxide synthase (T-NOS), Guanosine cyclase (GC), and Cyclic guanosine monophosphate (cGMP) in the penile tissue, as well as the expression of Endothelial nitric oxide synthase (eNOS) and Guanylate Cyclase Activator (GUCA) protein, in order to investigate the erectile function of the penis. Additionally, the quality of epididymal sperm was examined using an electron microscope. Furthermore, the serum sex hormone level and related protein expression were determined through the utilization of enzyme-linked immunosorbent assay and immunohistochemistry techniques. Pathological alterations and the ultrastructure of the testis were investigated using hematoxylin-eosin staining and transmission electron microscopy. Subsequently, the apoptosis of spermatogenic cells in the testes was assessed employing TUNEL, immunofluorescence, western blotting, and quantitative real-time polymerase chain reaction. ResultsThe administration of WSP has been found to enhance the behavioral performance and sexual behavior in aged mice. It's also could increase in serum levels of CR, UA, and BUN, as well as the elevation of SOD activity in kidney tissue, which subsequently leads to a reduction in MDA levels and an improvement in the structural damage caused by aging in the kidney tissue. Consequently, the renal function is enhanced. Additionally, WSP has been observed to elevate the levels of NO, T-NOS, GC, and cGMP in penile tissue, along with an increase in eNOS and GUCA protein expression, indicating an improvement in penile erectile function. The administration of WSP resulted in a decrease in the occurrence of programmed cell death in testicular germ cells, leading to an enhancement in sperm quality and the overall function of testicular spermatogenesis. This improvement can be attributed to the modulation of hormone levels and the regulation of SIRT1/3, p53, FOXO3, Bax, and Caspase-3 expression. ConclusionCollectively, our findings indicate that the administration of WSP has the potential to impede the occurrence of programmed cell death in testicular cells by modulating the expression of SIRT1/3 and subsequent genes associated with apoptosis. Consequently, this regulatory mechanism facilitates the proliferation of testicular cells and sustains the spermatogenic function of the testes. Consequently, by modulating the levels of sexual hormones in naturally aging mice, WSP ultimately enhances the quality of sperm and reproductive function. Concurrently, it also ameliorates age-related behavioral changes, renal function, and erectile function.

Proniosomal Gel-Loaded Phosphodiesterase Inhibitors (Sildenafil, Vardenafil, and Tadalafil): Prospects for Topical Penile Therapy of Tadalafil for Treatment of Erectile Dysfunction.

Oral phosphodiesterase inhibitors have emerged as a game changer for the treatment of erectile dysfunction (ED) since attaining FDA approval for its first member, sildenafil, in 1998. Topical penile therapy could be a viable replacement for oral medication that would transform the treatment of ED for many decades to come. This innovative idea could offer a safer topical alternative with less vision and cardiovascular side effects than the oral route. This work aims at developing proniosomal gels for three selected members (sildenafil, vardenafil, and tadalafil) and investigating the proniosomal gels on a rodent model. Niosomes derived from the parent proniosomal gels were characterized for entrapment efficiency (EE%), size, polydispersity index (PDI), zeta potential, and morphology. Proniosomal gels were evaluated for skin permeation, in vivo mating behaviors, and biochemical assays of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) post penile topical administrations. The optimized proniosomes loaded with tadalafil (F1-T) were compared with oral tablets (Cialis®). Proniosomal gels demonstrated significant enhancement of skin penetration by up to 5.5-fold, compared to control topical suspension. Tadalafil-loaded proniosomes showed superior skin permeability over sildenafil- and vardenafil-loaded proniosomes. In addition, significant improvement was noticed regarding intromission number, intromission ratio, NO, and cGMP for the proniosomal gel F1-T, compared to the untreated control. No statistically significant (p > 0.05) differences in sexual performance or biochemical parameters (NO and cGMP levels) were recorded among orally and topically (tadalafil proniosomal gel) administered groups. These findings support tadalafil topical penile therapy as a promising alternative to the oral route.

(016) The Effect of Phosphodiesterase-5-inhibitor on Female Sexual Function: A Systematic Review and Meta-analysis

Abstract Introduction The link between the attainment of healthy sexual function and a high quality of life in women is well established. However, while the management of male sexual dysfunction has been afforded huge research and development, far less time and effort has been lent in the achievement of successful remedies for females experiencing sexual dysfunction. The use of oral phosphodiesterase type 5 (PDE5) inhibitors, such as Sildenafil, has been the mainstay of treatment for male sexual dysfunction for the last two decades. PDE5 inhibitors have been shown to initiate penile erection, through smooth muscle relaxation and the subsequent increase in intracellular cGMP levels. PDE5-containing cells, similar to those found in the penis, have also been confirmed within the clitoris. No previous pooled analysis has been performed, looking at the possible effect of PDE5 inhibitors on female sexual function, in a population comprising of heterosexual cisgender population. Objectives We aimed to perform a systematic review and meta-analysis of the best possible available studies, to evaluate the efficacy and safety of PDE5 inhibitors versus placebo when used in an exclusively female population. Methods We conducted a comprehensive search of electronic databases (MEDLINE, PubMed, EMBASE, CINAHL, CENTRAL, SCOPUS, Google Scholar, Web of Science, EBSCO Host, TRIP and Epistemonikos), identifying studies comparing the PDE5 inhibitors versus placebo, and the resultant effect on female sexual function. The Cochrane risk-of-bias tool for RCTs and the Newcastle-Ottawa scale for observational studies were used to assess the included studies. Random effects modelling was used to calculate pooled outcome data. Results Searches of electronic databases resulted in 67 articles, from which 13 randomised control trials (RCTs) met the eligibility criteria, enrolling a total of 1605 patients. The use of PDE5 inhibitor was associated with a significantly improved orgasm satisfaction (P=0.01), increased sexual arousal (P=0.02), increase in sexual satisfaction (P=0.007) when compared to placebo. The use of PDE5i was also found to have a significant association with the incidence of headaches (P&amp;lt;0.0001) and flushing (P&amp;lt;0.00001). Conclusions Our meta-analysis demonstrated that the use PDE5 inhibitor was associated with a significantly higher attainment of orgasm satisfaction, sexual arousal, and sexual satisfaction when administered to a solely female population, when compared with placebo. However, the use of PDE5 inhibitors was also associated with a significantly increased risk of headache and flushing. Further large-scale high-quality RCTs are required in order to further evaluate the potential role of PDE5i’s in women, particularly in those suffering from significant sexual dysfunction. These future studies should focus on a more homogenous patient group, with stricter definitions of inclusion criteria. Disclosure

Hydrogen sulfide regulates the redox state of soluble guanylate cyclase in CSE-/- mice corpus cavernosum microcirculation

The corpus cavernosum (CC) is a highly vascularized tissue and represents an excellent example of microcirculation. Indeed, erectile dysfunction is considered an early index of cardiovascular disease. Hydrogen sulfide (H2S) at the vascular level is endogenously produced from L-cysteine mainly by the action of cystathionine-γ-lyase (CSE) and plays a role in CC vascular homeostasis. Here we have evaluated the involvement of the endogenous H2S in the regulation of the soluble guanylate cyclase (sCG) redox state. The lack of CSE-derived endogenous H2S, in CSE-/- mice, disrupted the eNOS/NO/sGC/PDE pathway. Indeed, the absence of CSE-derived endogenous H2S caused a significant reduction of the relaxant response to riociguat, an sGC redox-dependent stimulator. Conversely, the response to cinaciguat, an sGC redox-independent activator, was not modified. The relevance of the role played at the redox level of the endogenous H2S was confirmed by the findings that in CC harvested from CSE-/- mice there was a significant reduction of GCβ1 expression coupled with a decrease in CYP5R3, a reductase involved in the regulation of the redox state of sGC. These molecular changes driven by the lack of endogenous H2S translate into a significant reduction in cGMP levels. The replenishment of the lack of H2S with an H2S donor rescued the relaxant response to riociguat in CC of CSE-/- mice. In conclusion, the endogenous CSE-derived H2S plays a physiological key role in the regulation of the redox state of sGC in CC microcirculation.

Delta 9-tetrahydrocannabinol conserves cardiovascular functions in a rat model of endotoxemia: Involvement of endothelial molecular mechanisms and oxidative-nitrative stress.

In endotoxemic models, the inflammatory parameters are altered to a favorable direction as a response to activation of cannabinoid receptors 1 and 2. The phytocannabinoid Δ9-tetrahydrocannabinol (THC) is an agonist/partial antagonist of both cannabinoid receptors. This report targets the effects of THC on the cardiovascular system of endotoxemic rats. In our 24-hour endotoxemic rat model (E. coli derived lipopolysaccharide, LPS i.v. 5mg/kg) with THC treatment (LPS+THC 10 mg/kg i.p.), we investigated cardiac function by echocariography and endothelium-dependent relaxation of the thoracic aorta by isometric force measurement compared to vehicle controls. To evaluate the molecular mechanism, we measured endothelial NOS and COX-2 density by immunohistochemistry; and determined the levels of cGMP, the oxidative stress marker 4-hydroxynonenal, the nitrative stress marker 3-nitrotyrosine, and poly(ADP-ribose) polymers. A decrease in end-systolic and end-diastolic ventricular volumes in the LPS group was observed, which was absent in LPS+THC animals. Endothelium-dependent relaxation was worsened by LPS but not in the LPS+THC group. LPS administration decreased the abundance of cannabinoid receptors. Oxidative-nitrative stress markers showed an increment, and cGMP, eNOS staining showed a decrement in response to LPS. THC only decreased the oxidative-nitrative stress but had no effect on cGMP and eNOS density. COX-2 staining was reduced by THC. We hypothesize that the reduced diastolic filling in the LPS group is a consequence of vascular dysfunction, preventable by THC. The mechanism of action of THC is not based on its local effect on aortic NO homeostasis. The reduced oxidative-nitrative stress and the COX-2 suggest the activation of an anti-inflammatory pathway.

Pharmacokinetic effect of Astragalus membranaceus and Panax notoginseng saponins on arginine absorption and nitric oxide production in healthy subjects

Background: To the best of our knowledge, there are no clinical trials conducted with Astragalus and ginseng extracts on nitric oxide (NO) levels in humans. Therefore, this study aimed to examine whether the standardized intake of Astragalus membranaceus and Panax notoginseng saponins (APS) could increase NO production by enhancing arginine absorption and reducing levels of asymmetric dimethylarginine (AMDA).Methods: A clinical trial involving healthy adult participants aged between 20 to 80 years was conducted as a randomized, double-blind, cross-over study. The participants received 5 g of arginine powder and one capsule of APS or placebo twice, with a wash-out period between each administration. Plasma and urine were collected for testing, and 24 subjects were included for analysis after excluding six subjects with great individual differences.Results:It was found that after APS supplementation, the area under the curve (AUC) of arginine significantly increased by 17.3% (p = 0.041), the maximum concentration (Cmax) increased by 11.1%, and the Arg/ADMA ratio significantly increased by 167.1% (p = 0.007). Moreover, urinary nitrate and cGMP levels increased by 20.8% and 18.9%, respectively.Conclusions: APS showed increases in arginine absorption, decrease ADMA levels, and enhance NO production. With these findings, the addition of APS to arginine supplements could be advantageous for pre-workout and cardiovascular health.Keywords:Astragalus membranaceus and Panax notoginseng saponins (APS), arginine, asymmetric dimethylarginine (ADMA), nitric oxide (NO), cyclic guanosine monophosphate (cGMP)Clinical trial registration: NCT05024123

The Phosphoproteome of the Rd1 Mouse Retina, a Model of Inherited Photoreceptor Degeneration, Changes after Protein Kinase G Inhibition.

Retinitis pigmentosa (RP) is a frequent cause of blindness among the working population in industrial countries due to the inheritable death of photoreceptors. Though gene therapy was recently approved for mutations in the RPE65 gene, there is in general no effective treatment presently. Previously, abnormally high levels of cGMP and overactivation of its dependent protein kinase (PKG) have been suggested as causative for the fatal effects on photoreceptors, making it meaningful to explore the cGMP-PKG downstream signaling for more pathological insights and novel therapeutic target development purposes. Here, we manipulated the cGMP-PKG system in degenerating retinas from the rd1 mouse model pharmacologically via adding a PKG inhibitory cGMP-analogue to organotypic retinal explant cultures. A combination of phosphorylated peptide enrichment and mass spectrometry was then applied to study the cGMP-PKG-dependent phosphoproteome. We identified a host of novel potential cGMP-PKG downstream substrates and related kinases using this approach and selected the RAF1 protein, which may act as both a substrate and a kinase, for further validation. This showed that the RAS/RAF1/MAPK/ERK pathway may be involved in retinal degeneration in a yet unclarified mechanism, thus deserving further investigation in the future.

Uro-protective role of chrysin against cyclophosphamide-induced hemorrhagic cystitis in rats involving the turning-off NF-κB/P38-MAPK, NO/PARP-1 and STAT-3 signaling cascades

BackgroundChemotherapeutic agents are used to treat a wide range of cancer types, but they cause serious side effects which must be managed after treatment. Cyclophosphamide (CYP) is one of chemotherapeutic drugs that causes hemorrhagic cystitis (HC) induced by acrolein. ObjectiveThe current investigation intended to uncover the role of chrysin (CHR) in CYP-induced HC in rats and explore the signaling pathway beyond this effect. Analysisprocess: A single dose of CYP (200 mg/kg/IP) was injected, meanwhile CHR (25, 50 and 100 mg/kg, P.O) was administered respectively for 7 days prior to CYP administration and resume for 7 days afterwards. Urinary bladder tissue was then isolated from all rats to assess oxidative stress and inflammatory biomarkers. Moreover, histopathological examinations were performed. ResultsTreatment with CHR showed a marked alleviation in oxidative stress biomarkers induced by CYP. Furthermore, CHR treatment presented a dose-dependent boost in the anti-inflammatory; IL-10 levels and a drop in the pro-inflammatory biomarkers; IL-1β, IL-6, and TNF–α. Additionally, stabilization of the PARP-1 protein expression was also detected thus preventing DNA damage. Similarly, CHR restored the urinary bladder cGMP levels. Notably, CHR treatment was accompanied with inhibition in NF-κB/p38-MAPK, NO/PARP-1 and STAT-3 signaling pathways inflammatory cascades. All these findings conformed with the histopathological examinations as well as iNOS immunostaining in the urinary bladder tissue. ConclusionCo-administration of CHR and CYP attained uro-protective therapeutic potential to guard against HC as well as spot the tangled mechanism of CHR in attenuating the HC induced by CYP.

Erectile Dysfunction Treatment Using Stem Cell Delivery Patch in a Cavernous Nerve Injury Rat Model.

Erectile dysfunction (ED) is a common and feared complication of radical prostatectomy (RP) for prostate cancer. Recently, tissue engineering for post-prostatectomy ED has been attempted in which controlled interactions between cells, growth factors, and the extracellular matrix (ECM) are important for the structural integrity if nerve regeneration. In this study, we evaluated the effects of a biomechanical ECM patch on the morphology and behavior of human bone marrow-derived mesenchymal stem cells (hBMSCs) in a bilateral cavernous nerve injury (BCNI) rat model. The ECM patch, made of decellularized human fibroblast-derived ECM (hFDM) and a biocompatible polyvinyl alcohol (PVA) hydrogel, was tested with human bone marrow-derived mesenchymal stem cells (hBMSCs) on a bilateral cavernous nerve injury (BCNI) rat model. In vitro analysis showed that the hFDM/PVA + hBMSCs patches significantly increased neural development markers. In vivo experiments demonstrated that the rats treated with the hFDM/PVA patch had higher ICP/MAP ratios, higher ratios of smooth muscle to collagen, increased nNOS content, higher levels of eNOS protein expression, and higher cGMP levels compared to the BCNI group. These results indicate that the hFDM/PVA patch is effective in promoting angiogenesis, smooth muscle regeneration, and nitrergic nerve regeneration, which could contribute to improved erectile function in post-prostatectomy ED.

Co-expression of soluble guanylyl cyclase subunits and PDE5A shRNA to elevate cellular cGMP level: A potential gene therapy for myocardial cell death.

Genetic manipulation on the NO-sGC-cGMP pathway has been rarely achieved, partially due to complexity of the soluble guanylyl cyclase (sGC) enzyme. We aim to develop gene therapy directly targeting the pathway to circumvent cytotoxicity and tolerance after prolonged use of NO-donors and the insufficiency of PDE inhibitors. In this study, we constructed lentivirus vectors expressing GUCY1A3 and GUCY1B3 genes, which encoded the α1 and β1 subunits of soluble guanylyl cyclase (sGC), respectively, to enhance cGMP synthesis. We also constructed lentiviral vector harboring PDE5A shRNA to alleviate phosphodiesterase activity and cGMP degradation. Transductions of human HEK293 cells with the constructs were successful, as indicated by the fluorescent signal and altered gene expression produced by each vector. Overexpression of GUCY1A3 and GUCY1B3 resulted in increased sGC enzyme activity and elevated cGMP level in the cells. Expression of PDE5A shRNA resulted in decreased PDE5A expression and elevated cGMP level. Co-transduction of the three lentiviral vectors resulted in a more significant elevation of cGMP in HEK293 cells without obvious cytotoxicity. To the best of our knowledge, this is the first study to show that co-expression of exogenous subunits of the soluble guanylyl cyclase could form functional enzyme and increase cellular cGMP level in mammalian cells. Simultaneous expression of PDE5A shRNA could alleviate feedback up-regulation on PDE5A caused by cGMP elevation. Further studies are required to evaluate the effects of these constructs in vivo.

Inhibitory Effect of Jinkui Shenqi Pills on Glucocorticoid-Enhanced Axial Length Elongation in Experimentally Myopic Guinea Pigs.

To explore the underlying mechanism of inhibition by Jinkui Shenqi Pills (JKSQP) on glucocorticoid-enhanced axial length elongation in experimental lens-induced myopia (LIM) guinea pigs. Sixty 2-week old male guinea pigs were randomly divided into 4 groups with 15 guinea pigs in each group, according to the random numbers generated by SPSS software: control, LIM, saline and JKSQP groups. The control group includes animals with no treatment, while the guinea pigs in the other 3 groups received lens-induced myopization on the right eyes throughout the experiment (for 8 weeks). The saline and JKSQP groups were given daily intraperitoneal injections of 10 mg/kg hydrocortisone for 2 consecutive weeks at the same time, and then orally administered either saline or JKSQP [13.5 g/(kg•d) for 6 consecutive weeks. Body weight, anal temperature and animal appearance were observed and recorded to evaluate the GC-associated symptoms. The ocular parameters, including refraction and axial length, were measured by streak retinoscopy and A-scan ultrasonography, respectively. The levels of plasma hormones associated with the hypothalamic-pituitary-adrenal axis (HPAA), including free triiodothyronine, free thyroxine, estradiol and testosterone, were measured by radioimmunoassay, and cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate were measured by enzyme-linked immunosorbent assay. In addition, the mRNA and protein expressions of retinal amphiregulin (AREG) was measured by quantitative real-time polymerase chain reaction and Western blotting, respectively. JKSQP effectively increased body weight and anal temperature, improved animal appearance and suppressed axial length elongation in glucocorticoid-enhanced myopic guinea pigs with normalization of 4 HPAA-associated plasma hormones (all P<0.05). The plasma level of cAMP was significantly increased, whereas the plasma level of cGMP and the mRNA and protein expressions of retinal AREG were decreased after treatment with JKSQP (all P<0.05). JKSQP exhibited a significant inhibitory effect on axial length elongation with decreased expression of AREG in the retina, and normalized 4 HPAA-associated plasma hormones and the expression of cAMP and cGMP in GC-enhanced myopic guinea pigs.