Physiological Salt Solution Research Articles

Molybdenum – A biodegradable implant material for structural applications?

Molybdenum as a potentially new biodegradable material was investigated. Degradation behavior of commercially high purity molybdenum was observed in simulated physiological salt solutions (Kokubo's SBF with/without TRIS-HCl, Cu2+ addition and 0.9% NaCl solution). Potentiodynamic polarization, immersion mass loss and ion concentration measurements paired with REM/EDX analysis reveal gradual dissolution of molybdenum in the proper order of magnitude for stent application, associated with formation of thin, non-passivating corrosion products. The underlying corrosion mechanism is discussed as well as a comparison to literature data. However, formation of calcium phosphates (CaP) in SBF significantly decreases corrosion rates. In-situ polarization was found to be a potential way for overcoming this problem and simultaneously enhancing corrosion above the benchmark for a degradable stent material. Statement of SignificanceBiodegradable metals have the potential to overcome severe complications common to orthopedic and cardio-vascular implants. However, the need for a material with moderate and predictable degradation, high strength and toughness as well as MRI suitability must be satisfied. Molybdenum as potential new biodegradable material may just fulfill these requirements. An overall positive picture of molybdenum as an interesting alternative to recently discussed metallic biodegradable materials can be concluded from the herein presented results and from literature data, showing directions for future research on the topic.

L-arginase induces vascular dysfunction in old spontaneously hypertensive rats.

Aging is a major non-modifiable risk factor for hypertension. Changes in aging are similar to those seen in hypertension in the vasculature. Also, aging increases the vascular dysfunction that occurs in hypertension. L-arginase action reduces substrate (L-arginine) availability for the formation of nitric oxide (NO). This reduces the level of NO and leads to reduced vasodilation and ultimately, vascular dysfunction. This study examines the hypothesis that age-dependent vascular dysfunction in SHRs is mediated by arginase. Young (12-14 weeks) and old (11-12 months) male Wistar and spontaneously hypertensive rats (SHR) were used. Mean arterial pressure (MAP) was measured in the rats. They were then euthanized and mesenteric resistance arteries (MRAs) and thoracic aortae were excised and placed in ice-cold physiological salt solution (PSS). Arterial segments were either snap-frozen in liquid nitrogen and stored for immunoblotting studies or cut into 2mm rings for reactivity studies. Cumulative concentration-response curves to acetylcholine (Ach: 10-9 - 3x10-5M) and sodium nitroprusside (SNP: 10-12 - 3x10-5 M) were performed in the absence or presence (30-minute exposure) of L-arginase, 0.05U/ML (MRA) or 0.5U/ML (aorta). Vessels were pre-contracted with phenylephrine (PE; 3x10-6M). MAP increased during aging in the SHRs p<0.05 but not in the Wistar rats. Arginase impaired the endothelium-dependent relaxation responses of thoracic aortic and MRA arterial rings to Ach in the old Wistars and SHRs (Emax aorta: 29.42±2.19% vs 7.94±1.86%). Arginase also impaired endothelium-independent relaxation response to SNP in the old SHRs only (Emax aorta: 88.62±4.10% vs 31.45±10.61%). We also observed no differences in the serum arginase activity in the four groups of rats. On the contrary, arginase activity in the aortae of young Wistar rats was reduced compared to other groups. Arginase impairs both endothelium-dependent and -independent vasorelaxation responses, through the NO signaling pathway.

Prostanoid receptors (version 2019.5) in the IUPHAR/BPS Guide to Pharmacology Database

Prostanoid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Prostanoid Receptors [659]) are activated by the endogenous ligands prostaglandins PGD2, PGE1, PGE2 , PGF2α, PGH2, prostacyclin [PGI2] and thromboxane A2. Measurement of the potency of PGI2 and thromboxane A2 is hampered by their instability in physiological salt solution; they are often replaced by cicaprost and U46619, respectively, in receptor characterization studies.

Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels

BackgroundLocal injection of BaCl2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment.MethodsThe intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3–4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (Vm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50–75 μL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant.ResultsAddition of 1.2% BaCl2 to PSS depolarized myofibers from − 79 ± 3 mV to − 17 ± 7 mV with a corresponding rise in [Ca2+]i; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h.ConclusionsBaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.

Effects of ozone nano-bubble water on mucositis induced by cancer chemotherapy

No effective, reliable treatment for stomatitis associated with cancer therapy has been established. This study focused on the its effectiveness of ozone nano-bubble water (ONBW) for the treatment of chemotherapy-induced stomatitis. Oral mucositis was induced in 14-week-old male Sprague-Dawley rats (N = 21). The animals were randomly divided into 3 groups: 7 without treatment (control); 7 treated with physiological salt solution (saline); and 7 treated with ONBW. Animals were weighed on Days 7, 9, 11, and 16. Stomatitis grade evaluation and bacterial count measurements were performed before rinsing in all animals 3, 5, and 10 days after acetic acid irritation (Days 9, 11, and 17 respectively). Weight loss after stomatitis creation was observed in all groups, with significant differences between the control and ONBW groups and between the saline and ONBW groups on Day 16. The stomatitis grade did not worsen during the experimental period in any group, with the lowest grades in the ONBW group on Days 11 and 16. Significant differences were identified between the control and ONBW groups and between the saline and ONBW groups on Days 11 and 16. Oral bacterial counts tended to decrease over time in all three groups, with the greatest decrease in the ONBW group, followed by the saline group. The decrease in the bacterial count was steepest in the ONBW group. Rinsing out the oral cavity with ONBW decreased bacterial counts and encouraged the healing of oral chemotherapy-induced stomatitis. ONBW may be an effective treatment for chemotherapy-induced stomatitis.

How Well Can You Tailor the Charge of Lipid Vesicles?

Knowledge and control of surface charge or potential is important for tailoring colloidal interactions. In this work, we compare widely used zeta potential (ζ) measurements of charged lipid vesicle surface potential to direct measurements using the surface force apparatus (SFA). Our measurements show good agreement between the two techniques. On varying the fraction of anionic lipids dimyristoylphosphatidylserine (DMPS) or dimyristoylphosphatidylglycerol (DMPG) mixed with zwitterionic dimyristoylphosphatidylcholine (DMPC) from 0 to 100 mol % we observed a near-linear increase in membrane surface charge or potential up to 20-30 mol % charged lipids beyond which charge saturation occurred in physiological (high) salt conditions. Similarly, in low salt concentrations, a linear increase in charge/potential was found but only up to ∼5-10 mol % charged lipids beyond which the surface charge or potential leveled off. While a lower degree of ionization is expected due to the lower dielectric constant (ε ∼ 4) of the lipid acyl chain environment, increasing intramembrane electrostatic repulsion between neighboring charged lipid head groups at higher charge loading contributes to charge suppression. Measured potentials in physiological salt solutions were consistent with predictions using the Gouy-Chapman-Stern-Grahame (GCSG) model of the electrical double layer with Langmuir binding of counterions, but in low salt conditions, the model significantly overestimated the surface charge/potential. The much lower ionization in low salt (maximum ∼1-2% of total lipids ionized) instead was consistent with counterion condensation at the bilayer surface which limited the charge that could be obtained. The strong interplay between membrane composition, lipid headgroup ionization, electrolyte concentration, and solution pH complicates exact prediction and tuning of membrane surface charge for applications. However, the theoretical frameworks used here can provide guidelines to understand this interplay and establish a range of achievable potentials for a system and predict the response to triggers like pH and salt concentration changes.

Structure, transport properties, and dielectric properties of lactic acid/pyruvic acid aqueous solution in a biofuel cell: A molecular simulation study

Molecular dynamics (MD) simulation of lactic acid (L‐acid) and pyruvic acid (P‐acid) in a physiological salt solution has been performed, which is used as the electrolyte in a sort of biofuel cell working on a human skin. The structure, frequency distribution of molecules, dielectric properties, rotational correlation functions, and diffusion coefficients have been calculated. The anomalous behavior of dielectric constants for the lower concentration of L‐acid and P‐acid has been obtained from MD. The anomalous dielectric constant is discussed in relation with the various physical properties of the solution including the life time of the interaction in the solution. MD results suggest that the anomalous behavior is supposed to be attributed to the difference of interactions between ions in the lower and higher concentration region. POLYM. ENG. SCI., 59:2474–2478, 2019. © 2019 Society of Plastics Engineers

The effects of varying Mg2+ ion concentrations on contractions to the cotransmitters ATP and noradrenaline in the rat vas deferens.

The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg2+ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg2+ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg2+ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 μM and ATP 100 μM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 μM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg2+ 1.2 mM) as 100%, lowering Mg2+ to 0 mM enhanced the ATP peak to 170 ± 7% and raising Mg2+ to 3 mM decreased it to 39 ± 3%; the noradrenaline peak was not affected by lowering Mg2+ to 0 mM (97 ± 3%) but was decreased to 63 ± 4% in high Mg2+ (3 mM). Contractions to exogenous ATP, but not noradrenaline, were increased in Mg2+ 0 mM and both were inhibited with Mg2+ 3 mM. Changing Mg2+ concentration affects the contractions elicited by the cotransmitters ATP and noradrenaline. The greatest effects were to potentiate the contraction to ATP in Mg2+ 0 mM and to inhibit the contraction to both ATP and noradrenaline in high Mg2+. Future publications should clearly justify any decision to vary the magnesium concentration from normal (1.2 mM) values.

Prostanoid receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Prostanoid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Prostanoid Receptors [644]) are activated by the endogenous ligands prostaglandins PGD2, PGE1, PGE2 , PGF2α, PGH2, prostacyclin [PGI2] and thromboxane A2. Measurement of the potency of PGI2 and thromboxane A2 is hampered by their instability in physiological salt solution; they are often replaced by cicaprost and U46619, respectively, in receptor characterization studies.

Gastrointestinal and uterine smooth muscles relaxant and anti-inflammatory effects of corchorus olitorius leaf extract in laboratory animal models.

Corchorus olitorius is reportedly used in ethno-medicine to arrest threatened miscarriage and other conditions associated with excessive uterine contractions. The plant is also used as a purgative, demulscent and an anti-inflammatory agent. Against the background of ethno-medicinal use, this current work was designed to evaluate the gastrointestinal and uterine smooth muscles relaxant and anti-inflammatory effects of Corchorus olitorius leaf extract (COLE). Pieces of uterine and gastrointestinal tissues were suspended separately in organ baths containing ideal physiological salt solutions bubbled with air and were tested for responses to standard drugs and COLE, then repeated in the presence of antagonists. Anti-inflammatory study was carried out via the egg albumin-induced paw edema model in rats. The application of COLE to pieces of uterine tissue significantly decreased the amplitudes of contractions in a dose dependent manner such that the highest dose applied (666.67 μg/ml) achieved a 100% inhibitory effect. Oxytocin induced contractions were also significantly inhibited by both salbutamol and COLE. On the isolated rabbit jejunum, the effect of COLE was also inhibitory and like atropine, significantly inhibited acetylcholine induced contractions. In the in vivo study, the extract inhibited charcoal meal movement in test rats when compared with control. Anti-inflammatory effect of COLE was significant and compared favourably with that of aspirin following in vivo trials. COLE therefore, may be a good tocolytic, anti-diarrheal and anti-inflammatory agent and offers hope of new drug discovery for such uses.

Chemical composition, physical and chemical properties and biological value of turkey muscle and adipose tissues using biogenic stimulants

The effectiveness of the biogenic stimulants action based on the larvae of the SITR bees drone brood and adult individuals of the ST drones on the chemical composition, physical and chemical, commodity technological properties, nutritional and biological value of the muscle and fatty tissues in young turkeys is studied. The turkey-poults of the 1st group received injections of a physiological salt solution, and the 2nd and 3rd experimental groups, respectively, of the SITR and ST biological stimulants, three times at the age of 1, 7 and 14 days at a dose of 0.1 ml per 50 g of live weight. It was established that in the middle sample of the muscle tissue in turkeys of the 2nd and 3rd experimental groups there were less water and more dry matter and protein. Biogenic stimulants contributed to the synthesis of muscle tissue in turkeys of the 2nd and 3rd experimental groups, in which contained 1.79 (B&gt;0.999) and 1.31 (B&gt;0.99) absolute % more protein and 0.32 and 0.23 absolute % less fat than in the control group. In the muscle tissue of turkeys stimulated with biogenic preparations, there were more tryptophan by 1.63 and 0.84 mg% and by 0.92 and 0.54 mg% less hydroxyproline than in turkeys at the same age of the control group. The protein-quality index of muscle tissue was higher in turkeys of the 2nd and 3rd experimental groups by 3.08 and 1.72% than in the control. The first place in the overall assessment of meat and broth quality was taken by the turkeys from the 2nd experimental group; the second place was taken by the turkeys of the third experimental group. Their superiority over the control group was 0.151 and 0.109 points, respectively. The fatty tissue of turkeys in all experimental groups has good quality and high digestibility indices. It has in its composition a large amount of polyunsaturated fatty acids; this indicates its high biological value.

Identification and quantification of gold engineered nanomaterials and impaired fluid transfer across the rat placenta via ex vivo perfusion

Development and implementation of products incorporating nanoparticles are occurring at a rapid pace. These particles are widely utilized in domestic, occupational, and biomedical applications. Currently, it is unclear if pregnant women will be able to take advantage of the potential biomedical nanoproducts out of concerns associated with placental transfer and fetal interactions. We recently developed an ex vivo rat placental perfusion technique to allow for the evaluation of xenobiotic transfer and placental physiological perturbations. In this study, a segment of the uterine horn and associated placenta was isolated from pregnant (gestational day 20) Sprague-Dawley rats and placed into a modified pressure myography vessel chamber. The proximal and distal ends of the maternal uterine artery and the vessels of the umbilical cord were cannulated, secured, and perfused with physiological salt solution (PSS). The proximal uterine artery and umbilical artery were pressurized at 80 mmHg and 50 mmHg, respectively, to allow countercurrent flow through the placenta. After equilibration, a single 900 μL bolus dose of 20 nm gold engineered nanoparticles (Au-ENM) was introduced into the proximal maternal artery. Distal uterine and umbilical vein effluents were collected every 10 min for 180 min to measure placental fluid dynamics. The quantification of Au-ENM transfer was conducted via inductively coupled plasma mass spectrometry (ICP-MS). Overall, we were able to measure Au-ENM within uterine and umbilical effluent with 20 min of material infusion. This novel methodology may be widely incorporated into studies of pharmacology, toxicology, and placental physiology.

Mechanism of Spasmogenic Activity Stimulated by Aqueous Ethanolic Leaf Extract of Mucuna pruriens on Isolated Uterine Muscle of Albino Rats

Aims: To investigate the effect of aqueous ethanolic leaf extract of this medicinal plant on isolated uterine smooth muscle strips of the rat and to determine its mechanism of action.&#x0D; Study Design: Laboratory-experimental design was used in this study.&#x0D; Place and Duration of Study: The study was carried out in the Department of Veterinary Physiology and Biochemistry of Michael Okpara University of Agriculture, Umudike, Nigeria, Department of Pharmacology and Toxicology of the Faculty of Pharmacy, University of Nigeria, Nsukka, and the Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, Nigeria between June and October 2014.&#x0D; Methodology: Fresh leaves of Mucuna pruriens were identified and collected by a taxonomist from Nsukka, Nigeria. The leaves were then air dried and pulverized into powder. This was then subjected to cold extraction using petroleum ether (70-90) and 70% aqueous ethanol, after which the extract was left to dry at room temperature. Estrogenised uterine strips (12mm) were harvested from non-pregnant, sexually matured albino rats (180 g -250 g) and suspended in a 35ml organ bath containing Krebs’ physiological salt solution. The organ bath was connected to an isometric electronic force displacement transducer and a physiograph. Drugs such as Salbutamol, Isoprenaline, Adrenaline, Propranolol, Atipamezole and Prazosin were used as either agonists or antagonists to determine the mechanism of action of the extract. Atropine sulphate and Cyproheptadine were also used as test drugs. Concentrations of these drugs presented in the body of this work represent the final nutrient bath concentrations.&#x0D; Results: M. pruriens caused a dose -dependent increase in uterine muscle contraction with an EC50 of 0.88 mg/ml, n=4. The contraction was unaffected by atropine sulphate (0.042 µmol), but abolished by salbutamol (0.012-0.4 µmol), isoprenaline (0.06-0.23 µmol), and adrenaline (16 nmol). The uterine muscle contractions were enhanced by propranolol (1 µmol) in a dose- dependent manner. Prazosin (0.069-0.14 µmol) and atipamezole (3.3-13.7 nmol) were unable to abolish contractions stimulated by the extract. However, 0.2 µmol of cyproheptadine caused 80% suppression of the extract –induced uterine contraction&#x0D; Conclusion: It is concluded that aqueous ethanolic leaf extract of M. pruriens, has ability to cause uterine smooth muscle contraction hence, justifies its reported use traditionally as a uterine stimulant. This contraction is most likely exerted via the 5-HT receptor activation (activated by low concentrations of serotonin).

Impact of Green Vegetables and Green Smoothies on Enamel Mineral Composition In Vitro

Background: Recently, smoothies with a high vegetable content have become very popular, especially among young adults. Objectives: This in vitro study was conducted to determine the erosive potential of selected green vegetables and green smoothies on human dental enamel. Methods: A total of ten green smoothies and eight freshly mixed vegetables were chosen, and pH, calcium and oxalic acid contents were measured. The enamel specimens were obtained from wisdom teeth and incubated with 3 selected foods (spinach, parsley, or green smoothie) for 12 and 24 hours (N = 6). Control samples were incubated with a physiological salt solution. An electron probe microanalyzer (Jeol JXA 8900RL) was utilized to analyze the concentrations of calcium, phosphorus, magnesium and fluorine at various depths ranging from 5 to 50 µm within the enamel samples. A visual examination of the specimen was carried out with a digital microscope. Results: The green smoothies showed pH values ranging from 3.6 to 4.2, and the values of the mixed vegetables ranged from 5.6 to 6.3. Only in the green smoothie group could a time-dependent release of different minerals be observed. After 12 and 24 hours of incubation, a pronounced loss of calcium and phosphorus was found to a depth of 15 µm. Digital microscopy of the enamel surfaces confirmed these findings. Conclusions: In our in vitro study, incubation with the spinach and parsley showed no damage to the enamel specimens; in the case of the investigated smoothies, a mild loss of minerals to a depth of 25 µm was observed. It must be considered that numerous factors influence the enamel surface in vivo, translating these results to in vitro conditions must be done with great caution.

ROLE OF CHLORIDE AND ANO1 FOR THE CONTRACTILITY OF MESENTERIC SMALL ARTERIES IN RATS WITH STREPTOZOTOCIN INDUCED DIABETES

Diabetes is associated with altered contractile responses of small arteries. In the streptozotocin (STZ) induced rat model of diabetes enhanced contractility several groups have reported an increased contractility. In this study we tested the hypothesis that chloride is important for the enhanced contractility in the STZ rats.Diabetes was induced in 6 weeks old rats by a single intraperitoneal injection of 60 mg/kg STZ, control rats received vehicle injection. Eight weeks later blood glucose was significantly increased in the STZ rats compared to control rats (24.6±1.2 mmol/L and 6.7±1.1 mmol/L, STZ (n=9) and control (n=9), respectively). The rats were killed and mesenteric small arteries were isolated. The contractile response to increasing concentrations of norepinephrine of mesenteric small arteries was assessed in isometric myographs in physiological salt solution and in physiological salt solution where Cl− was substituted with aspartate. Expression of the Ca2+ activated Cl− channel ANO1 was assessed with qPCR and Western blotting in mesenteric small arteries.The maximal contraction to norepinephrine in physiological salt solution was higher in arteries from STZ rats compared to control rats (5.12±0.33 N/m and 4.07±0.24 N/m, STZ (n=14) and control (n=13), respectively). In Cl− free solutions the maximal response to norepinephrine was reduced and no difference between STZ and control rats was found (3.76±0.26 N/m and 3.23±0.22 N/m, STZ (n=14) and control (n=13), respectively). Furthermore the effect of Cl− free solution on the area under the NE concentration response curve was bigger in arteries from STZ rats (2.47±0.42 and 1.39±0.26 STZ (n=14) and control (n=13), respectively). Expression of ANO1 mRNA was higher in arteries from STZ rats (181±10% and 100±18%, STZ (n=4) and control (n=4), respectively). Expression of ANO1 protein was higher in arteries from STZ rats (195±24% and 100±18%, STZ (n=9) and control (n=8), respectively).This study provides evidence that the enhanced contractility of mesenteric arteries from STZ rats is dependent on the chloride gradient over the cell membrane, and suggests that this may be consequent to enhanced expression of the Ca2+ activated Cl− channel ANO1.Support or Funding InformationDanish Council for Independent Research (7016‐00013B)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chlorogenic Acid Increases Basal Tone with No Effect on Relaxation of Rat Ileal Smooth Muscles by Mechanisms Independent of PKG Mediated Effects on Myosin Light Chain Phosphorylation

Chlorogenic acid (CGA) is a dietary polyphenolic compound commonly present in coffee, fruits and vegetables. The pharmacological properties of CGA include antioxidant, antibacterial, anti‐inflammatory, and antithrombotic effects. Previous experimental studies have shown that CGA induces nitric oxide‐sGC‐PKG pathway to cause relaxation of the vascular smooth muscles. But the effects of CGA on ileal smooth muscles are still unknown. The present study is designed to test the effect of CGA on PKG mediated relaxation of rat ileal smooth muscle.Effects of CGA on basal tension and maximum contractile tension induced by physiological salt solution (PSS) containing 140 mM potassium in isolated rat ileal rings (3mm) were determined using tissue bath apparatus. CGA effects on myosin light chain phosphorylation at maximum contractile tension were also measured by freezing the ileal rings and extracting the protein and running Western blots. The role of PKG in regulating smooth muscle relaxation is tested by incubating ileal rings with specific PKG inhibitor Rp‐8‐Br‐cGMPS and measuring the basal tension, maximum contractile tension and myosin light chain phosphorylation as described above.The results show that basal tension increases from 0.27±0.1 (control group) to 0.91±0.3 and 0.87±0.2 (mV) when treated with CGA (10μM) and PKG inhibitor (4μM) respectively. The peak contractile tension in response depolarization by 140 mM K+ PSS changes from 100% (control group) to 101±12.2 % and 117±17.2 % when treated with CGA and PKG inhibitor respectively. The results from Western blots show that myosin light chain phosphorylation didn't change significantly between CGA (36.3±0.8 %) and PKG inhibitor (35.8±0.5 %) treatments.These results suggest that CGA is involved in increasing the basal tone of ileal smooth muscles when compared with the control group. In addition, CGA did not have any effect on the peak contractile tension and myosin light chain phosphorylation, suggesting that it may not be involved in ileal smooth muscle relaxation. However, PKG inhibitor in presence of CGA did increase basal and maximum contractile tension without any corresponding changes in myosin light chain phosphorylation indicating that other mechanisms that doesn't include PKG mediated effects on myosin light chain phosphorylation may be important in regulating basal tone of ileal smooth muscle by CGA.Support or Funding InformationSupported by internal grant of Cedarville University School of PharmacyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effect of Intermittent Oxytocin Pretreatment on Oxytocin-Induced Contractility of Human Myometrium In Vitro.

Prolonged continuous oxytocin administration during labor may induce oxytocin receptor desensitization, which attenuates the response of the myometrium to further oxytocin, increasing the risk of postpartum hemorrhage. The literature comparing pulsatile (intermittent) versus continuous oxytocin administration for induction and augmentation of labor is inconsistent with regard to maternal outcomes. We aimed to determine the effect of intermittent versus continuous oxytocin preexposure on myometrial responsiveness to subsequent oxytocin. We hypothesized that intermittent oxytocin pretreatment would result in superior subsequent oxytocin-induced contractility than continuous oxytocin pretreatment. This in vitro study was undertaken using myometrium obtained from women undergoing elective cesarean deliveries. Each myometrial strip was mounted in an individual organ bath with physiological salt solution under homeostatic conditions and allocated to one of 3 groups: (1) control (no pretreatment); (2) continuous (pretreatment with oxytocin 10(-5) M for 2 hours); or (3) intermittent (pretreatment with alternating oxytocin 10 M and physiological salt solution every 15 minutes, for 2 hours). After pretreatment, dose-response testing to oxytocin 10(-5) to 10(-5) M was performed and contractile parameters were measured. The primary outcome was motility index (MI, amplitude × frequency) of contractions. Eighteen women were recruited, and 86 successful experiments were performed (control n = 29, continuous n = 28, intermittent n = 29). The means (standard errors) of MI (√g·contractions/10 min) in the control, continuous, and intermittent groups were 2.34 (0.09), 1.78 (0.09), and 2.13 (0.11), respectively. The MI was significantly reduced in the continuous group when compared to the control (estimated difference [95% confidence interval {CI}], -0.56 [-0.81 to -0.31]; P < .01) and intermittent group (estimated difference [95% CI], -0.35 [-0.62 to -0.08]; P = .01). There was no significant difference in MI between the intermittent and control group (estimated difference [95% CI], -0.21 [-0.51 to 0.09]; P = .17). Human myometrium remains more responsive to subsequent oxytocin after intermittent compared to continuous exposure to oxytocin, most likely due to reduction in oxytocin receptor desensitization, or facilitation of receptor resensitization in the intermittent group. Hence, intermittent oxytocin administration during labor warrants further investigation as a technique to preserve uterine oxytocin responsiveness.

Red Blood Cells Specifically Bind a C‐peptide/Albumin Complex

People with Type 1 diabetes (T1D) require exogenous insulin due to the destruction of insulin‐secreting pancreatic β‐cells. However, even with insulin treatments, people with T1D develop complications such as retinopathy, neuropathy, and nephropathy that are often associated with poor microvascular blood flow. C‐peptide, a 31‐amino acid length peptide co‐secreted with insulin from the pancreatic β‐cells in a 1:1 mole ratio has been shown to improve microvascular blood flow in T1D rats and small‐scale human studies. C‐peptide indirectly enhances endothelium‐derived nitric oxide (NO) by stimulating the release of red blood cell (RBC) derived ATP, a recognized stimulus of endothelial NO synthase. Unfortunately, a receptor for C‐peptide has never been reported. Previously, using ELISA, we reported C‐peptide uptake by RBCs was approximately 1800 C‐peptide molecules/cell, but only when in the presence of albumin. In the absence of albumin, there was no measurable C‐peptide binding to the RBCs. We also report C‐peptide binding to albumin with a Kd of 5.7 ± (2.1) × 10−6 M; therefore, we hypothesize that C‐peptide binds to RBCs via a receptor that is specific for an albumin/Cpeptide complex. The binding of 99mTc labeled bovine serum albumin (BSA) to isolated RBCs, in the presence and absence of C‐peptide, was quantitatively determined by automatic gamma counting after repeated washing with a physiological salt solution. Sample counts per minute (CPM) were compared to a standard curve to determine the 99mTc‐BSA molecules per RBC. Results suggest BSA total binding to the RBCs saturates at approximately 1600 BSA molecules/RBC, whether or not C‐peptide is present, but that the BSA‐99mTc is specifically binding to RBCs in the presence of C‐peptide. Collectively, our data strongly suggest that the Cpeptide receptor has remained elusive because the receptor specifically binds an albumin/Cpeptide complex, as opposed to C‐peptide alone.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells

Our previous study found that the anthelmintic drug niclosamide relaxed the constricted arteries and inhibited proliferation and migration of vascular smooth muscle cells. Here, we investigated the effect of niclosamide ethanolamine (NEN) on trachea function and the proliferation and migration of trachea smooth muscle cells. Isometric tension of trachea was recorded by multi-channel myograph system. The cell proliferation was detected by using BrdU cell proliferation assay. The cell migration ability was evaluated by using scratch assay. The protein level was measured by using western blot technique. Acute treatment with NEN dose-dependently relaxed acetylcholine chloride (Ach)- and High K+ physiological salt solution (KPSS)-induced constriction of mice trachea. Pre-treatment with NEN inhibited Ach- and KPSS-induced constriction of mice trachea. NEN treatment inhibited proliferation of human bronchial smooth muscle cells (HBSMCs), inhibited migration of HBSMCs and rat primary trachea smooth muscle cells. NEN treatment activated adenosine monophosphate activated protein kinase (AMPK) activity and inhibited signal transducer and activator of transcription 3 (STAT3) activity in HBSMCs. In conclusion, niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells, indicating that niclosamide might be a potential drug for chronic asthma treatment.

Highly Adherent Parylene-C Coatings With Nanostructuring for Enhanced Cell Adhesion and Growth.

Parylene-C is a frequently used polymeric thin-film coating in medical applications and is known for its excellent biocompatibility and flexible deposition process. However, its use in long-term implants is limited due to its poor adhesion to metals in liquid environments. In this paper, we present a strategy to anchor Parylene-C to medical grade titanium (Ti) by means of nanostructuring the Ti substrates surface prior to Parylene coating. We observe that, after aging in the physiological salt solution for three days, Parylene coating lose their adhesion to bare titanium surfaces. However, the Parylene films deposited on nanostructured Ti surfaces retain full adhesion, even after aging them in the same solution for ten days. In addition, we demonstrate that combining nanostructured surfaces with very thin Parylene coatings provides the additional benefit of accelerating cell proliferation. Nanostructured surfaces showed cell proliferation without the typically required oxygen plasma treatment. Combining plasma treatment and nanostructuring further improved proliferation performance over smooth Parylene surfaces.