Blocker Of Ca2 Research Articles

Vasorelaxant effects and its mechanisms of the rhizome of Acorus gramineus on isolated rat thoracic aorta

In conventional medicine, the rhizome of Acorus gramineus Solander (AGR) is used to treat cardiovascular and cerebrovascular diseases. Decoctions containing AGR exert vasorelaxant effects. Therefore, this research aimed to delve deeper into the vasorelaxant effects and underlying mechanisms of AGR and its constituents (α-asarone and β-asarone). We assessed the vasorelaxant effect of a 50% ethanol extract of AGR (AGRE) using aortic rings from Sprague–Dawley rats pre-constricted with phenylephrine (PE) and potassium chloride (KCl). The findings suggested that the mechanism of this effect was independent of endothelial cells and was associated with vascular smooth muscle cells (VSMC). Since vasodilatory mechanisms associated with VSMC are predominantly influenced by K+ and Ca2+ channels, we explored various channels, including calcium-activated K+, voltage-dependent (delayed rectifier) K+, ATP-sensitive K+, inwardly rectifying K+, receptor-operated Ca2+ (ROCC), and voltage-dependent Ca2+ channels (VDCC). Selective blockers were used to examine K+ channels, which inhibited vasorelaxant effect of AGRE. These findings suggest that AGRE-induced vasorelaxation is facilitated through K+ channels. In addition, the blockage of Ca2+ influx was observed in both groups treated with PE and KCl. Therefore, AGR appears to block Ca2+ influx through ROCC and VDCC. In conclusion, AGR demonstrates vasorelaxant effects by acting on VSMC.

The vascular effects of peppermint (Mentha longifolia. L) on aorta in a mouse model: an ex-vivo and computational study

The present study examined the vascular effects of peppermint or mint (Mentha longifolia L.) using an abdominal aortic rings model. Concentration-response curves for mint oil were generated after precontracting isolated mouse aorta with phenylephrine. The effect of different receptor antagonists and ion channel or enzyme inhibitors on the vasorelaxant potential of mint oil were studied. Molecular docking studies were conducted using computational techniques to investigate the potential interactions between the bioactive constituents of mint oil and key vascular targets. The tension of aortic rings, which had been contracted by phenylephrine, relaxed as a function of the concentration of mint oil (0.0002–2 mg/mL). Pretreatment of the rings with the nitric oxide synthase inhibitor (L-NAME), a nonselective β-blocker (propranolol), and a muscarinic receptor blocker (atropine) didn’t show significant resistance to the vasodilatory effects of the mint oil. The vasodilatory effects of mint oil were significantly diminished when the rings were pretreated with glibenclamide, an inhibitor of ATP-sensitive K+ channels. In addition, indomethacin, a cyclooxygenase (COX) inhibitor, did influence mint oil’s tension in the preparations precontracted with phenylephrine. The present findings imply that ATP-sensitive K+ channels activation, blocking of Ca2+ channels, and inhibition of COX play a role in mediating the mint oil-induced vasorelaxation. Molecular docking studies of mint oil constituents showed that β-Elemene and Aromadendrene can interact with K+ and Ca2+ channels through various hydrophobic interactions with key amino acid residues. Additional work is needed to confirm the possible beneficial application of mint oil or its constituents in regulating the vascular tone.

Direct measurements of luminal Ca 2+ with endo-lysosomal GFP-aequorin reveal functional IP 3 receptors.

Endo-lysosomes are considered acidic Ca 2+ stores but direct measurements of luminal Ca 2+ within them are limited. Here we report that the Ca 2+ -sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca 2+ dynamics in this compartment. We show that Ca 2+ uptake is ATP-dependent and sensitive to blockers of endoplasmic reticulum Ca 2+ pumps. We find that the Ca 2+ mobilizing messenger IP 3 which typically targets the endoplasmic reticulum evokes robust luminal responses in wild type cells, but not in IP 3 receptor knock-out cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channel TRPML1. Stimulation with IP 3 -forming agonists also mobilized the store in intact cells. Super-resolution microscopy analysis confirmed the presence of IP 3 receptors within the endo-lysosomal system, both in live and fixed cells. Our data reveal a physiologically-relevant, IP 3 -sensitive store of Ca 2+ within the endo-lysosomal system.

Local Structure and Entropic Stabilization of Ca‐Based Molten Salt Electrolytes

AbstractHere molten salt electrolytes (MSEs) and specifically their physico‐chemical properties as a function of composition are reported on, with a special emphasis on the compositional entropy and targeting calcium battery application. By using MSEs, several problematic issues associated with organic electrolytes, such as the blocking of Ca2+ transfer at the electrode/electrolyte interfaces and electrolyte flammability, are avoided. Ca(FSI)2 salt in combination with the analogous Li‐, Na‐, and KFSI salts are used in equimolar compositions to first create several ternary MSEs, melting at (ca.) 60–75 °C, a melting temperature which is further reduced to (ca.) 55 °C for the unique quaternary MSE. This is ascribed to an increased entropy of mixing, which also contributes to an improved stability (re−)crystallization, as shown by Raman spectroscopy. Furthermore, molecular dynamics simulations of the quaternary MSE alongside density functional theory calculations targeting the ion‐ion interactions are used to elucidate the local structure in more detail, demonstrating that both the ionic radii and valence influence the coordination and solvation of the cations. These MSEs are stepping‐stones towards completely solvent‐free, semi‐solid, and ideally room‐temperature Ca‐conducting electrolytes.

Peroxiredoxin 1 inhibits streptozotocin-induced Alzheimer’s disease-like pathology in hippocampal neuronal cells via the blocking of Ca2+/Calpain/Cdk5-mediated mitochondrial fragmentation

Oxidative stress plays an essential role in the progression of Alzheimer’s disease (AD), the most common age-related neurodegenerative disorder. Streptozotocin (STZ)-induced abnormal brain insulin signaling and oxidative stress play crucial roles in the progression of Alzheimer’s disease (AD)-like pathology. Peroxiredoxins (Prxs) are associated with protection from neuronal death induced by oxidative stress. However, the molecular mechanisms underlying Prxs on STZ-induced progression of AD in the hippocampal neurons are not yet fully understood. Here, we evaluated whether Peroxiredoxin 1 (Prx1) affects STZ-induced AD-like pathology and cellular toxicity. Prx1 expression was increased by STZ treatment in the hippocampus cell line, HT-22 cells. We evaluated whether Prx1 affects STZ-induced HT-22 cells using overexpression. Prx1 successfully protected the forms of STZ-induced AD-like pathology, such as neuronal apoptosis, synaptic loss, and tau phosphorylation. Moreover, Prx1 suppressed the STZ-induced increase of mitochondrial dysfunction and fragmentation by down-regulating Drp1 phosphorylation and mitochondrial location. Prx1 plays a role in an upstream signal pathway of Drp1 phosphorylation, cyclin-dependent kinase 5 (Cdk5) by inhibiting the STZ-induced conversion of p35 to p25. We found that STZ-induced of intracellular Ca2+ accumulation was an important modulator of AD-like pathology progression by regulating Ca2+-mediated Calpain activation, and Prx1 down-regulated STZ-induced intracellular Ca2+ accumulation and Ca2+-mediated Calpain activation. Finally, we identified that Prx1 antioxidant capacity affected Ca2+/Calpain/Cdk5-mediated AD-like pathology progress. Therefore, these findings demonstrated that Prx1 is a key factor in STZ-induced hippocampal neuronal death through inhibition of Ca2+/Calpain/Cdk5-mediated mitochondrial dysfunction by protecting against oxidative stress.

Calorie restriction anti-hypertrophic effects are associated with improved mitochondrial content, blockage of Ca2+-induced mitochondrial damage, and lower reverse electron transport-mediated oxidative stress

Calorie restriction is a nutritional intervention that reproducibly protects against the maladaptive consequences of cardiovascular diseases. Pathological cardiac hypertrophy leads to cellular growth, dysfunction (with mitochondrial dysregulation), and oxidative stress. The mechanisms behind the cardiovascular protective effects of calorie restriction are still under investigation. In this study, we show that this dietetic intervention prevents cardiac protein elevation, avoids fetal gene reprogramming (atrial natriuretic peptide), and blocks the increase in heart weight per tibia length index (HW/TL) seen in isoproterenol-induced cardiac hypertrophy. Our findings suggest that calorie restriction inhibits cardiac pathological growth while also lowering mitochondrial reverse electron transport-induced hydrogen peroxide formation and improving mitochondrial content. Calorie restriction also attenuated the opening of the Ca2+-induced mitochondrial permeability transition pore. We also found that calorie restriction blocked the negative correlation of antioxidant enzymes (superoxide dimutase and glutatione peroxidase activity) and HW/TL, leading to the maintenance of protein sulphydryls and glutathione levels. Given the nature of isoproterenol-induced cardiac hypertrophy, we investigated whether calorie restriction could alter cardiac beta-adrenergic sensitivity. Using isolated rat hearts in a Langendorff system, we found that calorie restricted hearts have preserved beta-adrenergic signaling. In contrast, hypertrophic hearts (treated for seven days with isoproterenol) were insensitive to beta-adrenergic activation using isoproterenol (50 nM). Despite protecting against cardiac hypertrophy, calorie restriction did not alter the lack of responsiveness to isoproterenol in isolated hearts harvested from isoproterenol-treated rats. These results suggest (through a series of mitochondrial, oxidative stress, and cardiac hemodynamic studies) that calorie restriction possesses beneficial effects against hypertrophic cardiomyopathy.

Isothiazolinone Disrupts Reproductive Endocrinology by Targeting the G-Protein-Coupled Receptor Signaling.

The unintended exposure of humans and animals to isothiazolinones has led to an increasing concern regarding their health hazards. Isothiazolinones were previously found to disrupt reproductive endocrine homeostasis. However, the long-term reproductive toxicity and underlying mechanism remain unclear. In this study, life-cycle exposure of medaka to dichlorocthylisothiazolinone (DCOIT), a representative isothiazolinone, significantly stimulated the gonadotropin releasing hormone receptor (GnRHR)-mediated synthesis of follicle stimulating hormone and luteinizing hormone in the brain. Chem-Seq and proteome analyses revealed disturbances in the G-protein-coupled receptor, MAPK, and Ca2+ signaling cascades by DCOIT. The G protein αi subunit was identified as the binding target of DCOIT. Gαi bound by DCOIT had an enhanced affinity for the mitochondrial calcium uniporter, consequently changing Ca2+ subcellular compartmentalization. Stimulation of Ca2+ release from the endoplasmic reticulum and blockage of Ca2+ uptake into the mitochondria resulted in a considerably higher cytoplasmic Ca2+ concentration, which then activated the phosphorylation of MEK and ERK to dysregulate hormone synthesis. Overall, by comprehensively integrating in vivo, ex vivo, in silico, and in vitro evidence, this study proposes a new mode of endocrine disrupting toxicity based on isothiazolinones, which is expected to aid the risk assessment of the chemical library and favor the mechanism-driven design of safer alternatives.

Molten Salt Based High Entropy Electrolytes for Calcium Batteries

Calcium (Ca) is in principle a very attractive metal anode for rechargeable batteries due to the large abundance of calcium in the Earth’s crust, its low electrochemical potential, and its low cost1. Until rather recently2, however, stable plating and stripping of Ca and hence rechargeability of Ca-batteries was deemed impossible, due to the stable phases formed at the electrolyte/electrode interface, completely blocking Ca2+ transfer3.Since this pivotal breakthrough, Ca-batteries have attracted more interest, but they are still in their very infancy and new battery designs – such as using novel electrolyte concepts – are needed to achieve stable cycling of Ca metal anodes, and this preferably at room temperature.Here we report on the use of molten salt electrolytes, i.e. binary and multi-component systems of inorganic cations and anions, and more specifically on their physico-chemical and electrochemical properties as a function of composition with special emphasis on the compositional entropy. By using completely solvent-free electrolytes issues associated with organic electrolytes and solvents, such as the blocking of Ca2+ transfer and electrolyte flammability3, are avoided. Furthermore, they should/do not induce any large polarization, as opposed to ionic liquid-based electrolytes4.In more detail, using the Ca(FSI)2 salt in combination with the analogous Li-, Na-, and KFSI salts in equimolar compositions, all with melting temperatures (significantly) above 100 °C, we can create electrolytes melting at ca. 60-75 °C for the ternary systems, which is further reduced to ca. 55 °C for the quaternary system. These lowered melting temperatures are achieved by increased entropy of mixing, reducing the Gibbs free energy. This can for example be illustrated by Raman spectroscopy via comparing the spectra of “as cast” electrolytes with the spectra for the ones “aged” for one week (Figure 1). Here the FSI anion band at 772 cm-1 (νsS-N-S)5 shows that the electrolytes partially (re-)crystallize/micro-separate for the ternary systems, but stay more stable for the quaternary system, which we, at least partially, attribute to an entropy effect. Reference s M. E. Arroyo-De Dompablo, A. Ponrouch, P. Johansson, and M. R. Palacín, Chem . Rev., 120, 6331–6357 (2020).A. Ponrouch, C. Frontera, F. Bardé, and M. R. Palacín, Nat. Mater., 15, 169–172 (2016).D. Aurbach, R. Skaletsky, and Y. Gofer, J . Electrochem Soc., 138, 3536–3545 (1991).Q. Pang et al., Nature, 608, 704–711 (2022).K. Matsumoto, T. Oka, T. Nohira, and R. Hagiwara, Inorg . Chem ., 52, 568–576 (2013). Figure 1

In-phasic cytosolic-nuclear Ca2+ rhythms in suprachiasmatic nucleus neurons.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian clock in mammals. SCN neurons exhibit circadian Ca2+ rhythms in the cytosol, which is thought to act as a messenger linking the transcriptional/translational feedback loop (TTFL) and physiological activities. Transcriptional regulation occurs in the nucleus in the TTFL model, and Ca2+-dependent kinase regulates the clock gene transcription. However, the Ca2+ regulatory mechanisms between cytosol and nucleus as well as the ionic origin of Ca2+ rhythms remain unclear. In the present study, we monitored circadian-timescale Ca2+ dynamics in the nucleus and cytosol of SCN neurons at the single-cell and network levels. We observed robust nuclear Ca2+ rhythm in the same phase as the cytosolic rhythm in single SCN neurons and entire regions. Neuronal firing inhibition reduced the amplitude of both nuclear and cytosolic Ca2+ rhythms, whereas blocking of Ca2+ release from the endoplasmic reticulum (ER) via ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors had a minor effect on either Ca2+ rhythms. We conclude that the in-phasic circadian Ca2+ rhythms in the cytosol and nucleus are mainly driven by Ca2+ influx from the extracellular space, likely through the nuclear pore. It also raises the possibility that nuclear Ca2+ rhythms directly regulate transcription in situ.

Role of K+ and Ca2+ Channels in the Vasodilator Effects of Plectranthus barbatus (Brazilian Boldo) in Hypertensive Rats.

Plectranthus barbatus, popularly known as Brazilian boldo, is used in Brazilian folk medicine to treat cardiovascular disorders including hypertension. This study investigated the chemical profile by UFLC-DAD-MS and the relaxant effect by using an isolated organ bath of the hydroethanolic extract of P. barbatus (HEPB) leaves on the aorta of spontaneously hypertensive rats (SHR). A total of nineteen compounds were annotated from HEPB, and the main metabolite classes found were flavonoids, diterpenoids, cinnamic acid derivatives, and organic acids. The HEPB promoted an endothelium-dependent vasodilator effect (~100%; EC50 ~347.10 μg/mL). Incubation of L-NAME (a nonselective nitric oxide synthase inhibitor; EC50 ~417.20 μg/mL), ODQ (a selective inhibitor of the soluble guanylate cyclase enzyme; EC50 ~426.00 μg/mL), propranolol (a nonselective α-adrenergic receptor antagonist; EC50 ~448.90 μg/mL), or indomethacin (a nonselective cyclooxygenase enzyme inhibitor; EC50 ~398.70 μg/mL) could not significantly affect the relaxation evoked by HEPB. However, in the presence of atropine (a nonselective muscarinic receptor antagonist), there was a slight reduction in its vasorelaxant effect (EC50 ~476.40 μg/mL). The addition of tetraethylammonium (a blocker of Ca2+-activated K+ channels; EC50 ~611.60 μg/mL) or 4-aminopyridine (a voltage-dependent K+ channel blocker; EC50 ~380.50 μg/mL) significantly reduced the relaxation effect of the extract without the interference of glibenclamide (an ATP-sensitive K+ channel blocker; EC50 ~344.60 μg/mL) or barium chloride (an influx rectifying K+ channel blocker; EC50 ~360.80 μg/mL). The extract inhibited the contractile response against phenylephrine, CaCl2, KCl, or caffeine, similar to the results obtained with nifedipine (voltage-dependent calcium channel blocker). Together, the HEPB showed a vasorelaxant effect on the thoracic aorta of SHR, exclusively dependent on the endothelium with the participation of muscarinic receptors and K+ and Ca2+ channels.

#5443 ASSOCIATION OF SERUM CALCIUM-TO-MAGNESIUM RATIO WITH CARDIOVASCULAR AND CEREBRAL VASCULAR DISEASES IN DIALYSIS PATIENTS

Abstract Background and Aims Cardiovascular disease (CVD) and cerebral vascular accident (CVA) are the important complications and the leading causes of death in dialysis patients. High serum calcium (Ca) level promotes the clotting cascades and vascular calcification, which leads to CVD and CVA. Magnesium (Mg) is a well-known Ca blocker, inhibiting the formation of thrombin. An imbalance between serum Ca and Mg levels has been reported as an associated factor with CVD and mortality. This study investigated the associations of serum Ca-Mg ratio with CVD and CVA in dialysis patients. Method We conducted a cross-sectional analysis using baseline data from a multicenter prospective cohort for dialysis patients in South Korea. A total of 860 patients were divided into tertile groups according to serum Ca-Mg ratio levels. Patients with serum Ca-Mg ratio less than 3.28 were classified as the low group, and patients with serum Ca-Mg ratio more than 3.87 were classified as the high group, and the rest were classified as the middle group. Serum Ca level was calculated and applied as corrected Ca level considering serum albumin level. The associations of serum Ca-Mg ratio with prevalence of acute coronary syndrome (ACS), congestive heart failure, aortic artery calcification, and CVA were assessed. Results The average age of all patients was 59.1±12.1 years, and the average duration of dialysis was 7.9±5.9 years, and the duration of the low group was shorter than those of other groups. The high group had higher prevalence of peripheral vascular disease (P = 0.004). Serum albumin, phosphorus and Mg levels in the high group were lower than other groups (P<0.001). The prevalence of ACS showed higher in the high group than in other groups (P = 0.017). The prevalence of cerebral infarction showed higher in the high group than the low group (P = 0.016). In multivariable logistic regression analysis, higher Ca-Mg ratio was independently associated with the prevalence of ACS (Odds ratio 1.321, 95% confidential interval 1.009-1.728, P = 0.043) and cerebral infarction (Odds ratio 1.392, 95% confidential interval 1.002-1.896, P = 0.036). Conclusion A high Ca-Mg ratio was associated with the development of ACS and cerebral infarction. If the optimal cut-off of Ca-Mg ratio is determined, it can be used as a predictive marker for ACS and cerebral infarction.

Two-pore channel 1 and Ca2+ release-activated Ca2+ channels contribute to the acrosomal pH-dependent intracellular Ca2+ increase in mouse sperm.

The acrosome is a lysosome-related vesicular organelle located in the sperm head. The acrosomal reaction (AR) is an exocytic process mediated by Ca2+ and essential for mammalian fertilization. Recent findings support the importance of acrosomal alkalinization for the AR. Mibefradil (Mib) and NNC 55-0396 (NNC) are two amphipathic weak bases that block the sperm-specific Ca2+ channel (CatSper) and induce acrosomal pH (pHa ) increase by accumulating in the acrosomal lumen of mammalian sperm. This accumulation and pHa elevation increase the intracellular Ca2+ concentration ([Ca2+ ]i ) and trigger the AR by unknown mechanisms of Ca2+ transport. Here, we investigated the pathways associated with the pHa increase-induced Ca2+ signals using mouse sperm as a model. To address these questions, we used single-cell Ca2+ imaging, the lysosomotropic agent Gly-Phe-β-naphthylamide (GPN) and pharmacological tools. Our findings show that Mib and NNC increase pHa and release acrosomal Ca2+ without compromising acrosomal membrane integrity. Our GPN results indicate that the osmotic component does not significantly contribute to acrosomal Ca2+ release caused by pHa rise. Inhibition of two-pore channel1 (TPC1) channels reduced the [Ca2+ ]i increase stimulated by acrosomal alkalinization. In addition, blockage of Ca2+ release-activated Ca2+ (CRAC) channels diminished Ca2+ uptake triggered by pHa alkalinization. Finally, our findings contribute to understanding how pHa controls acrosomal Ca2+ efflux and extracellular Ca2+ entry during AR in mouse sperm. KEY POINTS: The acrosomal vesicle is a lysosome-related organelle located in the sperm head. The acrosome reaction (AR) is a highly regulated exocytic process mediated by Ca2+ , which is essential for fertilization. However, the molecular identity of Ca2+ transporters involved in the AR and their mechanisms to regulate Ca2+ fluxes are not fully understood. In mammalian sperm, acrosomal alkalinization induces intracellular Ca2+ concentration ([Ca2+ ]i ) increase and triggers the AR by unknown molecular mechanisms of Ca2+ transport. In this study, we explored the molecular mechanisms underlying Ca2+ signals caused by acrosomal alkalinization using mouse sperm as a model. TPC1 and CRAC channels contribute to [Ca2+ ]i elevation during acrosomal alkalinization. Our findings expand our understanding of how the acrosomal pH participates in the physiological induction of the AR.

MP27-06 BROMELAIN INDUCES RELAXATION OF HUMAN CORPUS CAVERNOSUM TISSUE IN VITRO INDEPENDENT NITRIC OXIDE-CGMP PATHWAY: POSSIBLE SIGNIFICANCE FOR ERECTILE DYSFUNCTION

You have accessJournal of UrologyCME1 Apr 2023MP27-06 BROMELAIN INDUCES RELAXATION OF HUMAN CORPUS CAVERNOSUM TISSUE IN VITRO INDEPENDENT NITRIC OXIDE-CGMP PATHWAY: POSSIBLE SIGNIFICANCE FOR ERECTILE DYSFUNCTION Serap Gur, Suresh C. Sikka, Asim B. Abdel-Mageed, Didem Yilmaz-Oral, Sudha Talwar, and Wayne J.G. Hellstrom Serap GurSerap Gur More articles by this author , Suresh C. SikkaSuresh C. Sikka More articles by this author , Asim B. Abdel-MageedAsim B. Abdel-Mageed More articles by this author , Didem Yilmaz-OralDidem Yilmaz-Oral More articles by this author , Sudha TalwarSudha Talwar More articles by this author , and Wayne J.G. HellstromWayne J.G. Hellstrom More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bromelain (BRM) is a mixture of proteolytic enzymes found in several parts of the pineapple (Ananas comosus) plant. BRM is widely administered for its well-recognized properties, mainly anti-inflammatory, antithrombotic and fibrinolytic, anticancer, and immunomodulatory. BRM gained universal acceptance as a phytotherapeutic agent due to its extensive dietary usage and lack of side effects; however, the complete molecular mechanism of action of BRM has not been completely identified. This exploratory study focuses on the erectile responses of human cavernosal strips on isometric tension measurements. In this study, we aimed to evaluate the effectiveness of (a) pure BRM (from pineapple stem, Sigma-Aldrich, St Louis, MO), (b) BRM dietary supplement (BG, Natural Brand Bromelain 500mg tablet, GNC, Pittsburgh, PA) and (c) fresh pineapple juice (PAJ) on human corpus cavernosum (HCC). METHODS: HCC tissues were obtained from men undergoing penile prosthesis implantation (n =8-10) and prepared for organ bath experiments as per our published procedures. BRM-induced relaxation response (10-8-10-3 M) was performed in vitro using HCC strips in an organ bath. The effects of inhibitors [NG-nitro-L-arginine methyl ester (L-NAME, 100µM), 1H-[1,2,4]-oxadiazolole-[4,3-a] quinoxalin-10one (ODQ, 30µM), and tetraethylammonium (TEA, 100 µM, blocker Ca2+ - activated K+channels)] on BRM-mediated relaxation responses were evaluated. The relaxation effects of BRM, BG, and PAJ on phenylephrine (Phe)-induced contraction were also compared. The protein expression of endothelial (e) and neuronal (n) nitric oxide synthase (NOS) and vascular endothelium growh factor (VEGF) were determined using Western blotting and immunohistochemistry. RESULTS: BRM inhibited Phe-evoked contractions in a dose-dependent manner. All agents induced relaxation of HCC (maximum response: BRM- 62.87 ± 6.23%, BG - 61.95 ± 4.17%, PAJ -55.68 ± 5.52%) after Phe-contraction. Inhibitors of nitric oxide (L-NAME) and soluble guanylate cyclase (ODQ) as well as TEA failed to affect the BRM-induced HCC relaxations. The relaxant responses of HCC strips were observed in the presence or the absence of BRM products. Furthermore, eNOS and nNOS protein levels were decreased and were not restored after BRM, while VEGF levels remained unaltered. CONCLUSIONS: These data suggest that BRM have the capacity to exert in vitro penile relaxations. The underlying mechanism of BRM is likely independent of the nitric oxide-cyclic guanosine monophosphate pathway. Overall, these in vitro studies suggested that consumption of BRM and its supplements may represent a novel strategy to prevent and treat erectile dysfunction in men, especially due to inflammatory issues. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e364 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Serap Gur More articles by this author Suresh C. Sikka More articles by this author Asim B. Abdel-Mageed More articles by this author Didem Yilmaz-Oral More articles by this author Sudha Talwar More articles by this author Wayne J.G. Hellstrom More articles by this author Expand All Advertisement PDF downloadLoading ...

Upregulation of Brain's Calcium Binding Proteins in Mitragynine Dependence: A Potential Cellular Mechanism to Addiction.

Background: Mitragyna speciosa Korth or Kratom is widely used traditionally for its medicinal values. The major alkaloid content of kratom leaves is mitragynine, which binds to opioid receptors to give opioid-like effects. This study aimed to analyse the brain proteome of animals that displayed addictive behaviors. Design and Methods: Six groups (n=6-8) of rats made up of negative control, positive control using morphine (10 mg/kg), and treatment groups at low (1mg/kg) and high doses of mitragynine (30 mg/kg) for 1 and 4 days. The rats' behaviors were evaluated and subsequently the rats' brains were harvested for proteomic analysis that was performed by using 2D gel electrophoresis and LC/MS/MS. Results: The rats developed physical dependence only on day 4 following morphine and mitragynine (1 and 30mg/kg) treatments. Among the proteins that were up-regulated in treatment groups were four calcium-binding proteins, namely calretinin, F-actin, annexin A3 and beta-centractin. Conclusions: Upregulation of calretinin acted as low Ca2+ buffering upon the blockage of Ca2+ ion channel by mitragynine in the brain, which subsequently caused a reduction of GABA released and inversely increased the dopamine secretions that contributed to dependence indicators.

TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy.

Background: Cisplatin is a widely used anti-tumor agent but its use is frequently limited by nephrotoxicity. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel which is generally viewed as a sensor of oxidative stress, and increasing evidence supports its link with autophagy, a critical process for organelle homeostasis. Methods: Cisplatin-induced cell injury and mitochondrial damage were both assessed in WT and Trpm2-knockout mice and primary cells. RNA sequencing, immunofluorescence staining, immunoblotting and flowcytometry were applied to interpret the mechanism of TRPM2 in cisplatin nephrotoxicity. Results: Knockout of TRPM2 exacerbates renal dysfunction, tubular injury and cell apoptosis in a model of acute kidney injury (AKI) induced by treatment with cisplatin. Cisplatin-caused tubular mitochondrial damage is aggravated in TRPM2-deficient mice and cells and, conversely, alleviated by treatment with Mito-TEMPO, a mitochondrial ROS scavenger. TRPM2 deficiency hinders cisplatin-induced autophagy via blockage of Ca2+ influx and subsequent up-regulation of AKT-mTOR signaling. Consistently, cisplatin-induced tubular mitochondrial damage, cell apoptosis and renal dysfunction in TRPM2-deficient mice are mitigated by treatment with a mTOR inhibitor. Conclusion: Our results suggest that the TRPM2 channel plays a protective role in cisplatin-induced AKI via modulating the Ca2+-AKT-mTOR signaling pathway and autophagy, providing novel insights into the pathogenesis of kidney injury.

PS-R04-6: A CASE OF MALIGNANT NEPHROSCLEROSIS WITHDREW FROM TEMPORARY HEMODIALYS

In the Case of AKI (acute renal injury) due to malignant hypertension, appropriate BP (Blood Pressure) management is important and sometimes requires hemodialysis. Here we report a 49-year-old female patient with malignant hypertension, AKI and acute pulmonary edema. She presented with severe hypertension (BP213/150 mmHg), high serum creatinine level (Cr12.74 mg/dL), congestive heart in chest radiographic findings and nerve head edema (Scheie: H4S3). There were no findings associated with renal artery stenosis. She was diagnosed as AKI due to malignant hypertension. Nicardipine continuous intravenous infusion was carried out for BP control and temporary hemodialysis had begun almost simultaneously. On Day 5 of admission, she had received oral Ca blocker and low dose of ACE inhibitor (temocapril). BP level remained stable thereafter. Nevertheless, oliguric renal failure lasted for several weeks. On Day 10 of admission, we performed renal biopsy and confirmed onion-skin-like intimal thickening and luminal narrowing findings. These were corresponded to malignant nephrosclerosis, not to chronic nephritic syndrome. We continued 2 times per week hemodialysis using the indwelling catheter after discharge. Her daily urinary output increased substantially day by day (over 1000 ml/day) and her serum creatinine level became less and less around Cr 4.0 mg/dL. This case withdrew temporary hemodialysis over a period of five months conclusively. In some cases, renal dysfunction is improved slowly in the long-term course by appropriate BP management. Accordingly, it is necessary to evaluate the possibility whether or not withdrawing from hemodialysis in the cases of AKI due to malignant hypertension.

Melatonin delays ABA-induced leaf senescence via H2 O2 -dependent calcium signalling.

Precocious leaf senescence can reduce crop yield and quality by limiting the growth stage. Melatonin has been shown to delay leaf senescence; however, the underlying mechanism remains obscure. Here, we show that melatonin offsets abscisic acid (ABA) to protect photosystem II and delay the senescence of attached old leaves under the light. Melatonin induced H2 O2 accumulationaccompanied by an upregulation of melon respiratory burst oxidase homologD (CmRBOHD) under ABA-induced stress. Both melatonin and H2 O2 induced the accumulation of cytoplasmic-free Ca2+ ([Ca2+ ]cyt ) in response to ABA, while blocking of Ca2+ influx channels attenuated melatonin- and H2 O2 -induced ABA tolerance. CmRBOHD overexpression induced [Ca2+ ]cyt accumulation and delayed leaf senescence, whereasdeletion of Arabidopsis AtRBOHD, a homologous gene of CmRBOHD, compromised the melatonin-induced [Ca2+ ]cyt accumulation and delay of leaf senescence in Arabidopsis under ABA stress. Furthermore, melatonin, H2 O2 and Ca2+ attenuated ABA-induced K+ efflux and subsequent cell death. CmRBOHD overexpression and AtRBOHD deletion alleviated and aggravated the ABA-induced K+ efflux, respectively. Taken together, our study unveils a new mechanism by which melatonin offsets ABA action to delay leaf senescence via RBOHD-dependent H2 O2 production that triggers [Ca2+ ]cyt accumulation and subsequently inhibits K+ efflux and delays cell death/leaf senescence in response to ABA.

Mechanisms of sympathoexcitation via P2Y6 receptors.

Many drugs used in cardiovascular therapy, such as angiotensin receptor antagonists and beta-blockers, may exert at least some of their actions through effects on the sympathetic nervous system, and this also holds true for e.g., P2Y12 antagonists. A new target at the horizon of cardiovascular drugs is the P2Y6 receptor which contributes to the development of arteriosclerosis and hypertension. To learn whether P2Y6 receptors in the sympathetic nervous system might contribute to actions of respective receptor ligands, responses of sympathetic neurons to P2Y6 receptor activation were analyzed in primary cell culture. UDP in a concentration dependent manner caused membrane depolarization and enhanced numbers of action potentials fired in response to current injections. The excitatory action was antagonized by the P2Y6 receptor antagonist MRS2578, but not by the P2Y2 antagonist AR-C118925XX. UDP raised intracellular Ca2+ in the same range of concentrations as it enhanced excitability and elicited inward currents under conditions that favor Cl- conductances, and these were reduced by a blocker of Ca2+-activated Cl- channels, CaCCInh-A01. In addition, UDP inhibited currents through KV7 channels. The increase in numbers of action potentials caused by UDP was not altered by the KV7 channel blocker linopirdine, but was enhanced in low extracellular Cl- and was reduced by CaCCInh-A01 and by an inhibitor of phospholipase C. Moreover, UDP enhanced release of previously incorporated [3H] noradrenaline, and this was augmented in low extracellular Cl- and by linopirdine, but attenuated by CaCCInh-A01. Together, these results reveal sympathoexcitatory actions of P2Y6 receptor activation involving Ca2+-activated Cl- channels.

Novel Oxime Synthesized from a Natural Product of Senecio nutans SCh. Bip. (Asteraceae) Enhances Vascular Relaxation in Rats by an Endothelium-Independent Mechanism.

Senecio nutans Sch. Bip. and its constituents are reported to have antihypertensive effects. We isolated metabolite–1, a natural compound from S. nutans (4-hydroxy-3-(isopenten-2-yl)-acetophenone), and synthesized novel oxime – 1 (4-hydroxy-3-(isopenten-2-yl)-acetophenoxime) to evaluate their effect on vascular reactivity. Compounds were purified (metabolite–1) or synthetized (oxime–1) and characterized using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). Using pharmacological agents such as phenylephrine (PE) and KCl (enhancing contraction), acetylcholine (ACh), L-NAME (nitric oxide (NO) and endothelial function), Bay K8644-induced CaV1.2 channel (calcium channel modulator), and isolated aortic rings in an organ bath setup, the possible mechanisms of vascular action were determined. Pre-incubation of aortic rings with 10−5 M oxime–1 significantly (p < 0.001) decreased the contractile response to 30 mM KCl. EC50 to KCl significantly (p < 0.01) increased in the presence of oxime–1 (37.72 ± 2.10 mM) compared to that obtained under control conditions (22.37 ± 1.40 mM). Oxime–1 significantly reduced (p < 0.001) the contractile response to different concentrations of PE (10−7 to 10−5 M) by a mechanism that decreases Cav1.2-mediated Ca2+ influx from the extracellular space and reduces Ca2+ release from intracellular stores. At a submaximal concentration (10−5 M), oxime–1 caused a significant relaxation in rat aorta even without vascular endothelium or after pre-incubate the tissue with L-NAME. Oxime–1 decreases the contractile response to PE by blunting the release of Ca2+ from intracellular stores and blocking of Ca2+ influx by channels. Metabolite–1 reduces the contractile response to KCl, apparently by reducing the plasma membrane depolarization and Ca2+ influx from the extracellular space. These acetophenone derivates from S. nutans (metabolite–1 and oxime–1) cause vasorelaxation through pathways involving an increase of the endothelial NO generation or a higher bioavailability, further highlighting that structural modification of naturally occurring metabolites can enhance their intended pharmacological functions.

Dual Inhibition of Phosphodiesterase and Ca++ Channels Explains the Medicinal Use of Balanites aegyptiaca (L.) in Hyperactive Gut Disorders.

The present study attempted to evaluate and rationalize the medicinal use of the methanolic extract of the fruits of Balanites aegyptiaca (B. aegyptiaca) in the treatment of hyperactive gut disorders. The in vivo, castor oil-induced diarrhea model in mice was followed to test its antidiarrheal effect. To test the antispasmodic effect and to explore its pharmacodynamic details, isolated small intestines (ileum) obtained from rats were selected to provide physiological conditions for the ex vivo assays. In the in vivo assays, the orally administered extract of B. aegyptiaca protected mice from diarrheal drops with resultant percent inhibitions of 40% and 80% at the respective doses of 200 mg/kg and 400 mg/kg, while the highest protection (100%) was observed with a positive control drug, loperamide, at 10 mg/kg. In the ileum, B. aegyptiaca produced an antispasmodic effect in a concentration-dependent manner by inhibiting the carbachol (CCh; 1 µM) and high K+ (80 mM)-evoked spasms with resultant EC50 values of 1.44 mg/mL (1.08–1.78) and 1.27 mg/mL (0.98–1.66), respectively. Papaverine, a known phosphodiesterase enzyme (PDE) inhibitor and blocker of Ca++ channels (CCB), also inhibited both CCh and high K+ induced contractions at comparable EC50 values of 8.72 µM (7.92–9.24) and 8.14 µM (7.62–8.84), respectively. Contrary to the extract and papaverine, verapamil showed distinctly higher potency in regard to inhibiting high K+, compared to CCh-evoked spasms that had EC50 values of 0.16 µM (0.13–0.261) and 2.54 µM (2.28–2.92), respectively. The inhibitory effects of B. aegyptiaca on PDE were further confirmed when the pre-incubated extract shifted the isoprenaline-mediated relaxation curves (CRCs) towards the left, similar to papaverine, whereas the CCB-like effect was confirmed when the pre-incubated tissues with B. aegyptiaca caused deflection in the Ca++ CRCs towards the right, constructed in Ca++ free medium with suppression of the maximum response. Thus, this study provides detailed, mechanistic support for the medicinal use of B. aegyptiaca in the treatment of hyperactive gut disorders.