Reduction Of Intracellular Calcium Research Articles

Abstract Tu145: Exploring Naltrindole’s hypercontracture attenuating effects during myocardial ischemia via a novel mechanism that produces cardioprotective effects in myocardial ischemia/reperfusion injury

Introduction: Previously, naltrindole (NTI) produced robust cardioprotective effects (e.g.~80% reduction of infarct size) in both ex-vivo (2.5-5µM) and in-vivo (3.75-7.5 mg/kg) rat myocardial ischemia-reperfusion (MIR) injury models. Additionally, we recently showed that NTI 100-200µM attenuated phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release by a novel mechanism of action (MOA) devoid of opioid receptors. Hypothesis: We hypothesize NTI’s novel MOA is a reduction of intracellular calcium (Ca 2+ ). Testing this hypothesis, we conducted isolated perfused MIR assays using KB-R7943(KB). KB is known to reduce intracellular Ca 2+ via inhibition of reverse mode Na + /Ca 2+ exchanger and, therefore, should attenuate myocardial ischemic hypercontracture, infarct size, and left ventricular end diastolic pressure (LVEDP). We predict that both NTI and KB will improve LVEDP and decrease infarct size compared to vehicle (dH 2 O) control and naloxone (NX) used as a negative control. Methods: Hearts were isolated from anesthetized male Sprague Dawley (SD) rats (~300g). We then infused NTI 1.25-5µM, KB 10-20µM, or NX 10µM for 5 min into perfused hearts, just prior to global I(30min)/R(45min). Cardiac left ventricular function was measured via a pressure transducer and infarct size using 1% triphenyltetrazolium chloride staining in the MIR assay. Data were analyzed using ANOVA and Fishers post-hoc analysis, with p<0.05 considered statistically significant. Results: MIR assay: NTI 5µM, 2.5µM, 1.25µM and KB 20 µM reduced infarct size (4.8±3%, n=5, p<0.05), (6.8±0.8%, n=6, p<0.05), (12.2±3%, n=5), and (2.9±2%, n=5, p<0.05) respectively compared to vehicle (14.1±2%, n=9) and NX 10µM controls (18±3%, n=3). NTI 5 µM and KB 20 µM had significantly improved LVEDP to (32.4±11%, n=5, p<0.05) and (22.8±4%, n=5, p<0.05) respectively compared to vehicle control (72.4±6%, n=9) and NX 10µM controls (54.2±8%, n=3) Conclusion: Both NTI (2.5µM, 5µM) and KB (20µM) reduced infarct size and improved LVEDP. The novel MOA of NTI may be mediated via a reduction in intracellular Ca 2+ during myocardial ischemia. The infarct reducing effect of NTI can benefit patients undergoing elective PCI, CABG, and transplant procedures.

Naltrindole attenuates irreversible Hypercontracture during Myocardial Ischemia by a Novel Mechanism and elicits robust cardioprotective effects in Myocardial Ischemia/Reperfusion Injury

Introduction: Previous studies have shown that naltrindole (NTI) produced robust cardioprotective effects (e.g.~80% reduction of infarct size) in both ex-vivo (2.5-5μM) and in-vivo (3.75-7.5 mg/kg) rat myocardial ischemia-reperfusion (MIR) injury models. We recently showed that NTI 100-200μM attenuated phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release by a novel mechanism of action (MOA) devoid of opioid receptors. We hypothesize NTI’s novel MOA is a reduction of intracellular calcium (Ca2+). To test this hypothesis, we conducted PMN SO release and isolated perfused MIR assays using KB-R7943(KB). KB is known to reduce intracellular Ca2+ via inhibition of reverse mode Na+/Ca2+ exchanger and therefore should attenuate PMN SO release, myocardial ischemic hypercontracture, infarct size, and left ventricular end diastolic pressure (LVEDP). Methods: PMNs and hearts isolated from anesthetized male Sprague Dawley (SD) rats (~400g SO release assay, ~300g for perfused hearts). PMN SO release was stimulated using PMA (100nM) ± NTI 10-200 μM, KB 5-20 μM, or vehicle (0.5% DMSO; controls) and measured spectrophotometrically as a change in absorbance @ 550nm over 390 secs and cell viability was determined using 0.2% Trypan blue exclusion. We then infused NTI 1.25-5μM, KB 10-20μM, or naloxone (NX; negative control) 10μM for 5 min into perfused hearts, just prior to global I (30min)/R(45min). Cardiac LV function was measured via a pressure transducer and infarct size using 1% triphenyltetrazolium chloride staining in MIR assay. Data were analyzed using ANOVA and Fischers post-hoc analysis, with p<0.05 considered statistically significant. Results: PMN SO release assay: KB 10 μM, KB 20 μM, NTI 100 μM (~3.75 mg/kg), and NTI 200 μM(~7.5 mg/kg) significantly attenuated PMA induced PMN SO release by ~40% (KB 10μM, n=7, p<0.05), ~50% ((KB 20μM, n=7, p<0.05), ~30% (NTI 100 μM, n=7, p<0.05), 75% (NTI 200μM, n=7, p<0.05) respectively when compared to untreated control (n=10). MIR assay: NTI 5μM, 2.5μM, 1.25μM and KB 20 μM reduced infarct size (4.8±3%, n=5, p<0.05), (6.8±0.8%, n=6, p<0.05), (12.2±3%, n=5), and (2.9±2%, n=5, p<0.05) respectively compared to vehicle (14.1±2%, n=9) and NX 10μM controls (18±3%, n=3). NTI 5 μM and KB 20 μM had significantly improved LVEDP to (32.4±11%, n=5, p<0.05) and (22.8±4%, n=5, p<0.05) respectively compared to vehicle control (72.4±6%, n=9) and NX 10μM controls(54.2±8%, n=3) Conclusion: NTI and KB showed concentration-dependent effects on reducing PMN SO release. Both NTI (2.5μM, 5μM) and KB (20μM) reduced infarct size and improved LVEDP. The novel MOA of NTI is most likely mediated via a reduction in intracellular Ca2+ and subsequent hypercontracture during myocardial ischemia. The infarct reducing effect of NTI can benefit patients who will undergo elective PCI, CABG, and transplants. Funding: This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, Division of Research, and the Center for Chronic Diseases of Aging and Young Therapeutics, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Poly-arginine-18 (R18) Confers Neuroprotection through Glutamate Receptor Modulation, Intracellular Calcium Reduction, and Preservation of Mitochondrial Function.

Recent studies have highlighted that a novel class of neuroprotective peptide, known as cationic arginine-rich peptides (CARPs), have intrinsic neuroprotective properties and are particularly effective anti-excitotoxic agents. As such, the present study investigated the mechanisms underlying the anti-excitotoxic properties of CARPs, using poly-arginine-18 (R18; 18-mer of arginine) as a representative peptide. Cortical neuronal cultures subjected to glutamic acid excitotoxicity were used to assess the effects of R18 on ionotropic glutamate receptor (iGluR)-mediated intracellular calcium influx, and its ability to reduce neuronal injury from raised intracellular calcium levels after inhibition of endoplasmic reticulum calcium uptake by thapsigargin. The results indicate that R18 significantly reduces calcium influx by suppressing iGluR overactivation, and results in preservation of mitochondrial membrane potential (ΔΨm) and ATP production, and reduced ROS generation. R18 also protected cortical neurons against thapsigargin-induced neurotoxicity, which indicates that the peptide helps maintain neuronal survival when intracellular calcium levels are elevated. Taken together, these findings provide important insight into the mechanisms of action of R18, supporting its potential application as a neuroprotective therapeutic for acute and chronic neurological disorders.

What happens to the unsuccessful spermatozoa?

To study apoptosis as a functional pathway in mature spermatozoa and apoptosis correlated to the acrosome reaction via the intracellular calcium concentration, semen samples from 27 healthy human donors were treated with inducers of apoptosis (betulinic acid, thapsigargin), inducers of the acrosome reaction (thapsigargin, calcium ionophore) or hydrogen peroxide to produce reactive oxygen species with and without prior incubation with a calcium chelator. Computer-assisted sperm analysis, flow cytometry, and transmission electron microscopy were performed to analyze changes in the acrosomal status and in apoptotic features. Betulinic acid, thapsigargin, and the calcium ionophore treatment resulted in an increased number of sperm cells with caspase 9 and caspase 3 activation, disrupted mitochondrial membrane potential, and a reacted acrosome. Sperm motility was decreased in all cases. Transmission electron analyses showed ultra-morphological changes, such as membrane integrity, membrane blebbing, the formation of head vacuoles, defects of the nuclear envelope, nuclear fragmentation, and the acrosome reaction. Acrosome reaction and apoptotic features decreased due to the reduction in intracellular calcium by the calcium chelator NP-EGTA, AM. Therefore, apoptotic cell death in acrosome-reacted sperm cells mediated by high intracellular calcium levels is possible.

Pinetonina<sup>TM</sup>, an Intranasally Administered Essential Oil Preparation, Is Effective in Decrease of Cortisol Levels and on the Glutamate Release Modulation

Background: Most anxiety complaints are treated with pharmacological measures involving barbiturates and benzodiazepines, in which they may end up causing tolerance and pharmacological dependence. Integrative approaches such as aromatherapy are used in addition to medications to improve sleep quality and reduce anxiety. Thus, PinetoninTM, a phytocomplex obtained from a blend of essential oils aims to aid in the symptoms of stress and anxiety. Methods: The cytotoxicity of PinetoninTM was evaluated MTT assay using fibroblasts and astrocytes showed reduction in the cell viability only at high concentrations. Evaluation of intracellular calcium and determination of residual glutamate in the supernatant of astrocyte cultures showed agonist action of dihydroxyphenylglycine (DHPG) increasing linearly the concentration of intracellular calcium and the glutamate levels in the supernatants of the cultures. On the other hand, cultures of astrocytes treated with PinetoninTM showed residual glutamate levels in the supernatants reducing proportionally, as well as, intracellular calcium reduction. The determination of salivary cortisol showed a significant reduction in salivary cortisol levels in the group that received PinetoninTM. The evaluation of the electroencephalogram in patients treated with PinetoninTM had a significant increase (P TM treated fibroblasts with control culture. When PinetoninTM and linole are administered in astrocytic cells, there was a reduction of the intracellular concentration of Ca2+ against a control group treated with DHPG agonist. The evaluation of salivary cortisol demonstrated a reduction when the patient group was treated with PinetoninTM by purchasing the results against the group of patients treated with saline. Reinforcing the relaxed state of that group, alpha waves have been increased and reductions in beta waves. Conclusion: The results obtained after intranasal administration of PinetoninTM suggest that this phytocomplex can help reduce the symptoms of stress and the better quality of life.

Formononetin ameliorates mast cell‑mediated allergic inflammation via inhibition of histamine release and production of pro‑inflammatory cytokines

Various allergic diseases cause allergic inflammation, which is mediated by mast cells. The current study investigated the anti-allergic inflammatory effects of formononetin and its mechanism of action in vitro using mast cells. Levels of histamine and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6, were measured to assess the effects of formononetin on allergic inflammation. The activation of intracellular calcium and nuclear factor (NF)-κB, as well as the activity of caspase-1, were assessed to determine the mechanism of action. It was determined that difference concentrations of formononetin (0.1, 1 and 10 µM) suppressed histamine release and secretion of TNF-α, IL-1β and IL-6. Further investigations indicated that the effects of formononetin were associated with a reduction of intracellular calcium, suppression of NF-κB activation and upstream IκKα phosphorylation and inhibition of caspase-1 activity. Therefore, the results of the current study demonstrated that formononetin ameliorated mast cell-mediated allergic inflammation.

Generation and phenotypic characterization of Pde1a mutant mice.

It has been proposed that a reduction in intracellular calcium causes an increase in intracellular cAMP and PKA activity through stimulation of calcium inhibitable adenylyl cyclase 6 and inhibition of phosphodiesterase 1 (PDE1), the main enzymes generating and degrading cAMP in the distal nephron and collecting duct, thus contributing to the development and progression of autosomal dominant polycystic kidney disease (ADPKD). In zebrafish pde1a depletion aggravates and overexpression ameliorates the cystic phenotype. To study the role of PDE1A in a mammalian system, we used a TALEN pair to Pde1a exon 7, targeting the histidine-aspartic acid dipeptide involved in ligating the active site Zn++ ion to generate two Pde1a null mouse lines. Pde1a mutants had a mild renal cystic disease and a urine concentrating defect (associated with upregulation of PDE4 activity and decreased protein kinase A dependent phosphorylation of aquaporin-2) on a wild-type genetic background and aggravated renal cystic disease on a Pkd2WS25/- background. Pde1a mutants additionally had lower aortic blood pressure and increased left ventricular (LV) ejection fraction, without a change in LV mass index, consistent with the high aortic and low cardiac expression of Pde1a in wild-type mice. These results support an important role of PDE1A in the renal pathogenesis of ADPKD and in the regulation of blood pressure.

Diospyros kaki calyx inhibits immediate-type hypersensitivity via the reduction of mast cell activation

Context: Diospyros kaki L. (Ebenaceae) fruit is widely distributed in Asia and is known to exert anti-inflammatory and antithrombotic effects. Objective: We evaluated the inhibitory effect of aqueous extract of D. kaki calyx (AEDKC) on mast cell-mediated immediate-type hypersensitivity and underlying mechanism of action. Materials and methods: For in vivo, ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) and immunoglobulin (Ig) E-mediated passive cutaneous anaphylaxis (PCA) models were used. In the ASA, AEDKC (1–100 mg/kg) was orally administered 3 times during 14 days. In the PCA, AEDKC was orally treated 1 h before the antigen challenge. The control drug dexamethasone was used to compare the effectiveness of AEDKC. For in vitro, IgE-stimulated RBL-2H3 cells and primary cultured peritoneal mast cells were used to determine the role of AEDKC (0.01–1 mg/mL). Results: Oral administration of AEDKC dose dependently suppressed rectal temperature decrease and increases in serum histamine, total IgE, OVA-specific IgE, and interleukin (IL)-4 in the ASA. In the PCA, AEDKC reduced Evans blue pigmentation. Compared to dexamethasone (10 mg/kg), AEDKC (100 mg/kg) showed similar inhibitory effects in vivo. AEDKC concentration dependently suppressed the release of histamine and β-hexosaminidase through the reduction of intracellular calcium in mast cells. In addition, AEDKC decreased the expression and secretion of tumour necrosis factor-α and IL-4 by the reduction of nuclear factor-κB. The inhibitory potential of AEDKC (1 mg/mL) was similar with dexamethasone (10 μM) in vitro. Conclusions: We suggest that AEDKC may be a potential candidate for the treatment of mast cell-mediated allergic diseases.

Involvement of transglutaminase 2 and voltage-gated potassium channels in cystamine vasodilatation in rat mesenteric small arteries.

Vasodilatation may contribute to the neuroprotective and vascular anti-remodelling effect of the tissue transglutaminase 2 (TG2) inhibitor cystamine. Here, we hypothesized that inhibition of TG2 followed by blockade of smooth muscle calcium entry and/or inhibition of Rho kinase underlies cystamine vasodilatation. We used rat mesenteric small arteries and RT-PCR, immunoblotting, and measurements of isometric wall tension, intracellular Ca(2+) ([Ca(2+)]i ), K(+) currents (patch clamp), and phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain, in our experiments. RT-PCR and immunoblotting revealed expression of TG2 in mesenteric small arteries. Cystamine concentration-dependently inhibited responses to phenylephrine, 5-HT and U46619 and for extracellular potassium. Selective inhibitors of TG2, LDN 27129 and T101, also inhibited phenylephrine contraction. An inhibitor of PLC suppressed cystamine relaxation. Cystamine relaxed and reduced [Ca(2+)]i in phenylephrine-contracted arteries. In potassium-contracted arteries, cystamine induced less relaxation without changing [Ca(2+)]i , and these relaxations were blocked by mitochondrial complex inhibitors. Blockers of Kv 7 channels, XE991 and linopirdine, inhibited cystamine relaxation and increases in voltage-dependent smooth muscle currents. Cystamine and the Rho kinase inhibitor Y27632 reduced basal MYPT1-Thr(855) phosphorylation, but only Y27632 reduced phenylephrine-induced increases in MYPT1-Thr(855) and myosin regulatory light chain phosphorylation. Cystamine induced vasodilatation by inhibition of receptor-coupled TG2, leading to opening of Kv channels and reduction of intracellular calcium, and by activation of a pathway sensitive to inhibitors of the mitochondrial complexes I and III. Both pathways may contribute to the antihypertensive and neuroprotective effect of cystamine.

SG-HQ2 inhibits mast cell-mediated allergic inflammation through suppression of histamine release and pro-inflammatory cytokines.

In this study, we investigated the effect of 3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide) (SG-HQ2), a synthetic analogue of gallic acid (3,4,5-trihydroxybenzoic acid), on the mast cell-mediated allergic inflammation and the possible mechanism of action. Mast cells play major roles in immunoglobulin E-mediated allergic responses by the release of histamine, lipid-derived mediators, and pro-inflammatory cytokines. We previously reported the potential effects of gallic acid using allergic inflammation models. For incremental research, we synthesized the SG-HQ2 by the modification of functional groups from gallic acid. SG-HQ2 attenuated histamine release by the reduction of intracellular calcium in human mast cells and primary peritoneal mast cells. The inhibitory efficacy of SG-HQ2 was similar with gallic acid. Enhanced expression of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, interleukin-4, and interleukin-6 in activated mast cells was significantly diminished by SG-HQ2 100 times lower concentration of gallic acid. This inhibitory effect was mediated by the reduction of nuclear factor-κB. In animal models, SG-HQ2 inhibited compound 48/80-induced serum histamine release and immunoglobulin E-mediated local allergic reaction, passive cutaneous anaphylaxis. Our results indicate that SG-HQ2, an analogue of gallic acid, might be a possible therapeutic candidate for mast cell-mediated allergic inflammatory diseases through suppression of histamine release and pro-inflammatory cytokines.

A novel calcium-dependent mechanism of acquired resistance to IGF-1 receptor inhibition in prostate cancer cells.

Inhibition of the mitogenic insulin-like growth factor receptor 1 (IGF-1R) signaling axis is a compelling treatment strategy for prostate cancer. Combining the IGF-1R inhibitor ganitumab (formerly AMG 479) with standard of care androgen-deprivation therapy greatly delays prostate cancer recurrence in xenograft models; however, a significant proportion of these tumors ultimately acquire resistance to ganitumab. Here we describe the development of a stable and reproducible ganitumab-resistant VCaP human prostate cancer cell derivative termed VCaP/GanR to investigate the mechanism of acquired resistance to IGF-1R inhibition. Unlike parental VCaP, VCaP/GanR did not undergo apoptosis following ganitumab treatment. VCaP/GanR did not express increased levels of IGF-1R, insulin receptor, or phospho-AKT compared to parental VCaP. VCaP/GanR exhibited increased levels of phospho-S6 indicative of increased mTOR activity. However, acquired resistance to ganitumab was not dependent on increased mTOR activity in VCaP/GanR. Phospho-proteomic arrays revealed alterations in several calcium-regulated signaling components in VCaP/GanR compared to VCaP. Reduction of intracellular calcium using cell-permeable calcium-specific chelators restored ganitumab sensitivity to VCaP/GanR through inhibition of cell-cycle progression. These data suggest a new mechanism of resistance to IGF-1R inhibition involving calcium-mediated proliferation effects. Such pathways should be considered in future clinical studies of IGF-1R inhibitors in prostate cancer.

Curcumin attenuates fructose-induced vascular dysfunction of isolated rat thoracic aorta rings

Context: Consumption of high fructose is associated with metabolic abnormalities, insulin resistance, and hypertension. It is not known whether this hypertensive effect of fructose is related to metabolic abnormalities or due to the direct effect of fructose on blood vessels.Objective: Here, we investigated the direct effect of fructose on rat isolated aorta and the possible protective effect of curcumin.Materials and methods: The isolated rat thoracic aorta rings were used to measure the contractile responses to different concentrations of both phenylephrine and KCl, and the relaxant response to acetylcholine (Ach). The effect of curcumin (1 µM) alone or in combination with tempol (1 mM), a superoxide dismutase mimetic agent, and N-{[3(amino-methyl)-phenyl]-methyl} ethanimidamide dihydrochloride (1400 W), a specific inducible nitric oxide synthase (iNOS) inhibitor, (1 µM) on fructose-treated aorta was compared. The aortic rings were incubated with different treatments for 60 min before starting the experiment. Changes in the intracellular calcium in response to KCl and nitric oxide levels were also measured.Results and discussion: Fructose strongly increased the contractile response of aortic rings to both phenylephrine and KCl (Emax was increased by 147.3% and 150.5%, respectively) but it did not affect the relaxant response to Ach. Curcumin significantly decreased the hyper responsiveness of arterial rings to both vasopressors (for phenylephrine, Emax decreased from 147.3% in fructose incubated aorta to 81%, and for KCl, Emax decreased from 150.5% in fructose-incubated aorta to 77.24% respectively). Curcumin also reduces the intracellular calcium level (85% reduction in intracellular calcium). A 1400 W was the only agent that potentiates the effect of curcumin.Conclusion: Fructose has a direct deleterious effect on aortic vascular reactivity. Curcumin can partially protect against fructose-induced impairment in vascular contractility via an antioxidant effect and reduction of elevated intracellular calcium.

Galangin attenuates mast cell-mediated allergic inflammation

A great number of people are suffering from allergic inflammatory disease such as asthma, atopic dermatitis, and sinusitis. Therefore discovery of drugs for the treatment of these diseases is an important subject in human health. In this study, we investigated anti-allergic inflammatory effect of galangin and underlying mechanisms of action using in vitro and in vivo models. Galangin inhibited histamine release by the reduction of intracellular calcium in phorbol 12-mystate 13-acetate plus calcium ionophore A23187-stimulated human mast cells (HMC-1). Galangin decreased expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-8. The inhibitory effect of galangin on theses pro-inflammatory cytokines was related with c-Jun N-terminal kinases, and p38 mitogen-activated protein kinase, nuclear factor-κB, and caspase-1. Furthermore, galangin attenuated IgE-mediated passive cutaneous anaphylaxis and the expression of histamine receptor 1 at the inflamed tissue. The inhibitory effects of galangin were more potent than cromolyn, a known anti-allergic drug. Our results showed that galangin down-regulates mast cell-derived allergic inflammatory reactions by blocking histamine release and expression of pro-inflammatory cytokines. In light of in vitro and in vivo anti-allergic inflammatory effects, galangin could be a beneficial anti-allergic inflammatory agent.

P20 Glucocorticoids suppress hydrogen sulfide production in liver and kidneys

Chronic exposure to high glucocorticoid levels is often associated with alterations in the haemostatic system, arterial hypertension and atherosclerosis. Increased frequency of both thromboembolic events and high blood pressure has been reported in patients with Cushing’s syndrome. From the other side, recent research has shown that the endogenous gas hydrogen sulphide (H 2 S) induces relaxation of vessels through a direct effect on KATP-channels causing hyperpolarization and closure of voltage-dependent Ca 2+ -channels followed by a reduction in intracellular calcium as well as through the release of endothelium-derived hyperpolarizing factor and nitric oxide from the endothelium. Therefore, we hypothesized that probably the cardiovascular complications occurring in hypercorticism are determined at least partially by the decrease in H 2 S production.Wistar rats were treated with saline, Dexamethasone (Dex) (4 mg/kg/d intraperitoneally for 2 weeks) or Dex + Pyridoxine. Pyridoxine (10 mg/kg/d intraperitoneally) was administered every day during glucocorticoid treatment. Concentration of H 2 S in blood plasma was measured by the spectrophotometric methylene-blue method. The activities of cystathionine- γ -lyase (CSE), cysteine aminotransferase (CAT) and cystathionine- β -synthase (CBS) were measured in the homogenates of liver and kidneys. Rats were euthanized on the 8th and 15th days after the beginning of Dex administration.In general, in both terms of experiment Dex caused almost a 2-fold decrease in H 2 S blood plasma concentration compared to the control. Analysis of the time-dependent effect of Dex on the functioning of H 2 S producing enzymes showed the significant inhibition of CSE, CAT and CBS activities. CSEis expressed abundantly in mammalian cardiovascular system but it also appears to be the main H 2 S-forming enzyme in the liver and kidneys. After Dex injection for 7 days the liver CSE activity decreased in 1.2 times, whereas administration of hormone for 14 days resulted in the decrease of this index in 1.5 times. Another enzyme that has been reported to generate H 2 S in rat liver is CAT. We observed 1.2- and 1.6-fold decrease of CAT activity in liver after Dex administration for 7 and 14 days correspondingly. The CSE and CAT activities in kidneys of rats with hyperglucocorticoidemia did not change significantly compared with the control rats. CBS expression is significant mainly in the brain and it is not so considerable in other tissues. Despite this fact in our experiments we showed that in rats treated with Dex the significant inhibition of CBS activity occurs in both liver and kidneys on 8th and 15th days of experiment. Pyridoxine partially reversed both inhibition of CSE, CAT and CBS activities and H 2 S concentration in blood of rats treated with Dex.Thus, our results clearly demonstrated that Dex reduces H 2 S production in liver and kidneys and H 2 S level in blood. This allows to assume that reduced endogenous H 2 S production in the diseases associated with chronic increase of blood glucocorticoids concentration may play a role in the development of hypertension, atherosclerosis, heart ischemia and administration of Pyridoxine or H 2 S donors may be beneficial in prevention of such cardiovascular complications.

Preventing β-Cell Loss and Diabetes With Calcium Channel Blockers

Although loss of functional β-cell mass is a hallmark of diabetes, no treatment approaches that halt this process are currently available. We recently identified thioredoxin-interacting protein (TXNIP) as an attractive target in this regard. Glucose and diabetes upregulate β-cell TXNIP expression, and TXNIP overexpression induces β-cell apoptosis. In contrast, genetic ablation of TXNIP promotes endogenous β-cell survival and prevents streptozotocin (STZ)- and obesity-induced diabetes. Finding an oral medication that could inhibit β-cell TXNIP expression would therefore represent a major breakthrough. We were surprised to discover that calcium channel blockers inhibited TXNIP expression in INS-1 cells and human islets and that orally administered verapamil reduced TXNIP expression and β-cell apoptosis, enhanced endogenous insulin levels, and rescued mice from STZ-induced diabetes. Verapamil also promoted β-cell survival and improved glucose homeostasis and insulin sensitivity in BTBR ob/ob mice. Our data further suggest that this verapamil-mediated TXNIP repression is conferred by reduction of intracellular calcium, inhibition of calcineurin signaling, and nuclear exclusion and decreased binding of carbohydrate response element–binding protein to the E-box repeat in the TXNIP promoter. Thus, for the first time, we have identified an oral medication that can inhibit proapoptotic β-cell TXNIP expression, enhance β-cell survival and function, and prevent and even improve overt diabetes.

Ripe fruit of Rubus coreanus inhibits mast cell-mediated allergic inflammation

In this study, we investigated the effect of a water extract of the ripe fruits of Rubus coreanus Miq. (Rosaceae) (RFRC) on mast cell-mediated allergic inflammation and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases such as anaphylaxis, rhinitis, asthma and atopic dermatitis. RFRC dose-dependently inhibited compound 48/80-induced systemic anaphylaxis and serum histamine release in mice. RFRC reduced the immunoglobulin E (IgE)-mediated local allergic reaction, passive cutaneous anaphylaxis. RFRC attenuated histamine release from rat peritoneal mast cells and human mast cells by the reduction of intracellular calcium. RFRC decreased the phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187 (PMACI)-stimulated expression and secretion of pro-inflammatory cytokines in human mast cells. The inhibitory effect of RFRC on cytokine production was nuclear factor (NF)-κB- and mitogen-activated protein kinase (MAPK)-dependent. In addition, RFRC suppressed the activation of caspase-1. Our findings provide evidence that RFRC inhibits mast cell-derived allergic inflammatory reactions, and for the involvement of calcium, NF-κB, MAPKs and caspase-1 in these effects. Furthermore, in vivo and in vitro anti-allergic inflammatory effects of RFRC provide affirmative proof of a possible therapeutic application of this agent in allergic inflammatory diseases.

The Role of Endogenous H2S in Cardiovascular Physiology

Recent research has shown that the endogenous gas hydrogen sulphide (H2S) is a signalling molecule of considerable biological potential and has been suggested to be involved in a vast number of physiological processes. In the vascular system, H2S is synthesized from cysteine by cystathionine-γ-lyase (CSE) in smooth muscle cells (SMC) and 3- mercaptopyruvate sulfuresterase (3MST) and CSE in the endothelial cells. In pulmonary and systemic arteries, H2S induces relaxation and/or contraction dependent on the concentration of H2S, type of vessel and species. H2S relaxes SMC through a direct effect on KATP-channels or Kv-channels causing hyperpolarization and closure of voltage-dependent Ca2+-channels followed by a reduction in intracellular calcium. H2S also relaxes SMC through the release of endothelium- derived hyperpolarizing factor (EDHF) and nitric oxide (NO) from the endothelium. H2S contracts SMC through a reduction in nitric oxide (NO) availability by reacting with NO forming a nitrosothiol compound and through an inhibitory effect on endothelial nitric oxide synthase (eNOS) as well as a reduction in SMC cyclic AMP concentration. Evidence supports a role for H2S in oxygen sensing. Furthermore, reduced endogenous H2S production may also play a role in ischemic heart diseases and hypertension, and treatment with H2S donors and cysteine analogues may be beneficial in treatment of cardiovascular disease.

Effects of Gekko sulfated polysaccharide–protein complex on human hepatoma SMMC-7721 cells: Inhibition of proliferation and migration

Aim of the study Gekko swinhonis Guenther has been used as an anti-cancer drug in traditional Chinese medicine for hundreds of years. Here we investigated the structural characterization and anti-cancer effects of sulfated polysaccharide–protein complex (GSPP) isolated from Gekko swinhonis Guenther. Materials and methods The structure of GSPP was characterized by high performance liquid chromatography, gas chromatography, gas chromatography–mass spectrometry, β-elimination reaction, and NMR spectroscopy. SMMC-7721 cells were used to assess the influence of GSPP on hepatocellular carcinoma. Cell proliferation and survival was determined by trypan blue exclusion assay. Cell migration was performed by wound-healing and transwell assay. The secretion of IL-8 was detected by an enzyme-linked immunosorbent assay kit. Flow cytometry was used to analyze intracellular calcium concentration, as well as cell cycle distribution and apoptosis. Confocal microscopy was used to assess the localization and configuration of actin filaments. Results GSPP was chemically characterized as a sulfated polysaccharide–protein complex with O-glycopeptide linkages. Our results showed that GSPP inhibited the proliferation of SMMC-7721 cells and blocked cells in the S phase. No direct toxicity against cells was observed. Furthermore, GSPP inhibited the migration of SMMC-7721 cells with the reduction of intracellular calcium. Actin filaments were polymerized and accumulated in the cytoplasm of the treated cells, whereas the secretion of IL-8 was not significantly changed after GSPP exposure. Conclusion We describe an identified sulfated polysaccharide–protein complex, and demonstrate its direct effect on hepatocellular carcinoma cell migration via calcium-mediated regulation of the actin cytoskeleton reorganization.

Vasodilator efficacy of nitric oxide depends on mechanisms of intracellular calcium mobilization in mouse aortic smooth muscle cells

Reduction of intracellular calcium ([Ca(2+)](i)) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca(2+) mobilization during SMC contraction influenced NO efficacy. Isometric contractions by depolarization (high potassium, K(+)) or alpha-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca(2+)](i) (Fura-2) were measured in aortic segments from C57Bl6 mice. Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K(+) solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca(2+)](i), which was suppressed by sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K(+)-stimulated segments did not parallel a decline of [Ca(2+)](i). However, stimulation (BAY K8644) of L-type Ca(2+) influx diminished, while inhibition (nifedipine, 1-100 nM) augmented the relaxing capacity of NO. In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca(2+) re-uptake into the SR and was prominent when intracellular Ca(2+) was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca(2+)](i), presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca(2+) influx, and L-type Ca(2+) channel blockers increase the vasodilator efficacy of NO.

Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20)

Changes in intracellular calcium are necessary for the successful progression of mitosis in many cells. Both elevation and reduction in intracellular calcium can disrupt mitosis by mechanisms that remain ill defined. In this study we explore the role of transmembrane voltage-gated calcium channels (CaV channels) as regulators of mitosis in the mouse corticotroph cell line (AtT-20). We report that the nifedipine-sensitive isoform CaV1.2 is localized to the “poleward side” of kinetechores during metaphase and at the midbody during cytokinesis. A second nifedipine-sensitive isoform, CaV1.3, is present at the mid-spindle zone in telophase, but is also seen at the midbody. Nifedipine reduces the rate of cell proliferation, and, utilizing time-lapse microscopy, we show that this is due to a block at the prometaphase stage of the cell cycle. Using Fluo-4 we detect calcium fluxes at sites corresponding to the mid-spindle zone and the midbody region. Another calcium dye, Fura PE3/AM, causes an inhibition of mitosis prior to anaphase that we attribute to a chelation of intracellular calcium. Our results demonstrate a novel, isoform-specific localization of CaV1 channels during cell division and suggest a possible role for these channels in the calcium-dependent events underlying mitotic progression in pituitary corticotrophs.