Krebs Solution Research Articles

Endothelium-dependent relaxation mechanisms involve nitric oxide and prostanoids in the isolated bovine digital vein.

Endothelial dysfunction contributes to the development of ungulate's laminitis. Although extensively studied in equines, the endothelial function is not fully examined in bovine digital veins (BDVs). BDVs were studied under isometric conditions to describe the acetylcholine (ACh) endothelium-dependent relaxation. Concentration-response curves were constructed to phenylephrine, ACh, and sodium nitroprusside (SNP). Relaxation responses were evaluated using either phenylephrine or depolarizing high-potassium Krebs solution (DKS) as precontraction agents. Endothelium denudation and incubation with L-NAME (300μM), indomethacin (10μM) or both were used to explore endothelial-mediated mechanisms. Endothelium denudation did not modify phenylephrine and SNP responses, however, significantly (p<0.05) converted a relaxation (63.2±5%) response to ACh into a contraction (30.3±9%). The ACh-evoked relaxation was significantly (p<0.05) reduced in the presence of indomethacin (37.5±6%) and L-NAME (6.40±2%). The presence of both inhibitors abolished the ACh-evoked relaxation. Although DKS caused a higher precontraction than phenylephrine, ACh-evoked relaxation (22.4±3.4%) was still observed and was reduced by the combination of inhibitors (7.0±1.0%). The ACh endothelium-dependent relaxation in BDVs is essentially mediated by nitric oxide and endothelium-derived prostanoids. The BDV endothelium function is a dynamic component in the control of the bovine digital blood flow, particularly under endothelial dysfunction conditions when venoconstriction might lead to postcapillary resistance increase.

Placental growth factor blunts uterine artery responses to angiotensin II.

Changes in maternal serum concentration of placental growth factor (PlGF) and vascular response to intravascular infusion of Angiotensin II (Ang II) follow a bell-shaped curve pattern during gestation. This study evaluates the effects of PlGF and soluble vascular endothelial growth factor receptor-1 (sFlt-1) on responses of human uterine arteries (UA) to Ang II. Experimental. Baylor College of Medicine and Texas Children's Hospital-Pavilion for Women. Uterine arteries samples (n=14) were obtained from normotensive women undergoing caesarean hysterectomy at≥32weeks. Uterine arteries rings were incubated with (1) Krebs solution; (2) PlGF at 1.45, 14.5, and 500pg/ml; (3) sFlt-1 at 2ng/ml; and (4) a combination of sFlt-1, and PlGF. Dose-contraction responses to Ang II were determined in UA rings incubated in the above-mentioned conditions. Responses were also measured in presence of L-NAME or inhibitors of endothelium-derived hyperpolarising factor: apamine and charybdotoxin. The t-test was used for comparisons. Changes in vascular reactivity of UA rings. PlGF blunted (P=0.03) and sFlt-1 increased (P<0.01) the UA maximum responses to Ang II. A combination of sFlt-1 and PlGF blunted UA responses to Ang II (P<0.05). l-NAME, apamine, and charybdotoxin reversed the relaxation effects of PlGF on UA responses to Ang II (P<0.05). PlGF contributes to the blunted vascular response to Angiotensin II during normotensive pregnancies and sFlt-1 appears to attenuate this effect. PlGF and sFlt-1 may contribute to the regulation of vascular tone during pregnancy by altering the vascular response to Angiotensin II. Baylor College of Medicine. Placental growth factor and soluble vascular endothelial growth factor receptor-1 modulate the uterine artery response to Angiotensin II in normotensive pregnant women.

Thermodynamic assessment of the use of alternative metabolic energy resources on work performance efficiency of spontaneous rat myometrium contractions

Uterine smooth muscles (myometrium) are employed during labour to expel foetus (infant) out of the uterus. Exergy efficiency of the rat myometrium muscle contractions were assessed with the data collected in organ bath experiments in Krebs solutions. The major energy source, glucose, of the Krebs solution was substituted with sucrose, sodium pyruvate and calcium and lactate to assess the effect of the metabolic energy source on the exergy efficiency of work performance. Force versus time graphs were correlated with the work done during the contractions. Muscle contraction exergy efficiency was calculated as the ratio of work done by a muscle strip to the total exergy supplied. Exergy efficiency of the contractions was the highest, 36.7%, when lactate was the metabolic energy source and it was followed by 35.3% when pyruvate was the metabolic energy source. Exergy efficiencies of the cases with glucose and sucrose were 34.1% and 23.5%, respectively. The findings of this study may open way to further studies which may eventually help women give birth to babies with less pain by increasing exergy efficiency of the process.

Lipid emulsion attenuates the vasodilation induced by a toxic dose of a calcium channel blocker through its partitioning into the lipid phase.

The present in vitro study examined whether lipid emulsion attenuates the vasodilation evoked by toxic doses of calcium channel blockers (bepridil, verapamil, nifedipine and diltiazem) via their partitioning into the lipid phase. The effects of the calcium channel blockers alone, the lipid emulsion and calcium channel blocker mixture, and the centrifuged aqueous extract (CAE) obtained from ultracentrifugation of the lipid emulsion and calcium channel blocker mixture on isolated endothelium-denuded rat aortas precontracted with phenylephrine were observed. The effects of lipid emulsion on calcium channel blocker concentration in the Krebs solution were examined using ultraperformance liquid chromatography. A mixture of lipid emulsion with either bepridil or verapamil and the corresponding CAE more effectively attenuated vasodilation than either bepridil or verapamil alone, whereas the vasodilation induced by the mixture of lipid emulsion and either bepridil or verapamil was not significantly different from that induced by the corresponding CAE. The magnitude of the lipid emulsion-mediated reduction in vasodilation and calcium channel blocker concentration was as follows: bepridil > verapamil > nifedipine or diltiazem. These results suggest that lipid emulsion attenuates vasodilation induced by a toxic dose of bepridil and verapamil, seemingly through partitioning of the calcium channel blocker into the lipid phase.

Impact of ginsenoside-Rg3 on catecholamine secretion in the perfused model of the rat adrenal medullae

Background: The present study was the first attempt to explore the characteristics of ginsenoside-Rg3 (Rg3) on release of catecholamines (CA) in the perfused rat adrenal medullae and also to verify the underlying action mechanism. Materials and Methods: The adrenal medulla was separated by some modification of the previous method and perfused with Krebs solution. CA was assayed directly by the fluorometry. Results: Rg3 reduced acetylcholine (ACh)-produced CA release in a dose- and time-dependent manner. Rg3 time-dependently depressed CA release produced by 3-(m-chloro-phenyl-carbamoyl-oxy)-2-butynyltrimethyl ammonium chloride (McN-A-343), 1,1-dimethyl-4-phenyl piperazinium iodide, and angiotensin II. In the presence of Rg3, the CA release produced by high K+, veratridine, cyclopiazonic acid, and methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4- (2-trifluoromethyl-phenyl)-pyridine-5-carboxylate (Bay-K-8644) was also markedly suppressed. However, during the simultaneous perfusion of Rg3 and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), the release of CA produced by ACh, angiotensin II, Bay-K-8644, and veratridine was restored closely to the level of each control, in contrast to that of Rg3-treatment alone. The nitric oxide (NO) release was significantly elevated by Rg3-treatment. Furthermore, in the coexistence of Rg3 and fimasartan, ACh-produced CA release was more significantly reduced as compared to that of fimasartan-treatment alone. Conclusions: We present the first evidence that Rg3 markedly depresses the CA secretion produced by activation of neuronal cholinergic and angiotensinergic receptors. Rg3-produced inhibition appears to be evoked not only by blocking the inflow of Na+ and Ca2+ into adrenomedullary cells but also by preventing the Ca2+ release from intracellular storage, partly through enhancement of NO release by NO synthase activation. Coadministration of Rg3 and fimasartan may be clinically beneficial for the treatment of cardiovascular diseases. Abbreviations used: Rg3: Ginsenoside-Rg3; VDCC: L-type voltage-dependent Ca2+ channels; Bay–K-8644: Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl-phenyl)-pyridine-5-carboxylate; ACh: Acetylcholine; McN-A-343: 3-(m-chloro-phenyl-carbamoyl-oxy)-2-butynyltrimethyl ammonium; DMPP: 1.1-dimethyl-4-phenyl piperazinium; NO: Nitric oxide; L-NAME: Nω-nitro-l-arginine methyl ester; nNOS: Neuronal NO synthase.

Thermodynamic assessment of the use of alternative metabolic energy resources on work performance efficiency of spontaneous rat myometrium contractions

Uterine smooth muscles (myometrium) are employed during labour to expel foetus (infant) out of the uterus. Exergy efficiency of the rat myometrium muscle contractions were assessed with the data collected in organ bath experiments in Krebs solutions. The major energy source, glucose, of the Krebs solution was substituted with sucrose, sodium pyruvate and calcium and lactate to assess the effect of the metabolic energy source on the exergy efficiency of work performance. Force versus time graphs were correlated with the work done during the contractions. Muscle contraction exergy efficiency was calculated as the ratio of work done by a muscle strip to the total exergy supplied. Exergy efficiency of the contractions was the highest, 36.7%, when lactate was the metabolic energy source and it was followed by 35.3% when pyruvate was the metabolic energy source. Exergy efficiencies of the cases with glucose and sucrose were 34.1% and 23.5%, respectively. The findings of this study may open way to further studies which may eventually help women give birth to babies with less pain by increasing exergy efficiency of the process.

Loss of inhibitory synapses causes locomotor network dysfunction of the rat spinal cord during prolonged maintenance in vitro

The isolated spinal cord of the neonatal rat is widely employed to clarify the basic mechanisms of network development or the early phase of degeneration after injury. Nevertheless, this preparation survives in Krebs solution up to 24 h only, making it desirable to explore approaches to extend its survival for longitudinal studies. The present report shows that culturing the spinal cord in oxygenated enriched Basal Medium Eagle (BME) provided excellent preservation of neurons (including motoneurons), glia and primary afferents (including dorsal root ganglia) for up to 72 h. Using DMEM medium was unsuccessful. Novel characteristics of spinal networks emerged with strong spontaneous activity, and deficit in fictive locomotion patterns with stereotypically slow cycles. Staining with markers for synaptic proteins synapsin 1 and synaptophysin showed thoroughly weaker signal after 3 days in vitro. Immunohistochemical staining of markers for glutamatergic and glycinergic neurons indicated significant reduction of the latter. Likewise, there was lower expression of the GABA-synthesizing enzyme GAD65. Thus, malfunction of locomotor networks appeared related to loss of inhibitory synapses. This phenomenon did not occur in analogous opossum preparations of the spinal cord kept in vitro. In conclusion, despite histological data suggesting that cultured spinal cords were undamaged (except for inhibitory biomarkers), electrophysiological data revealed important functional impairment. Thus, the downregulation of inhibitory synapses may account for the progressive hyperexcitability of rat spinal networks despite apparently normal histological appearance. Our observations may help to understand the basis of certain delayed effects of spinal injury like chronic pain and spasticity.

Antiasthmatic effect of Curcuma aeruginosa extract on isolated organ of the trachea

Background:Asthma is a major health problem worldwide. Antiasthma drugs have side effects and can be expensive. It is important to develop antiasthma drugs from medicinal plants that have fewer side effects and are cheaper. One of the medicinal plants used for antiasthma treatment comes fromCurcuma aeruginosa(Zingiberaceaefamily). The aim of the research is to examine spasmolytic activity of ethanol extract ofC. aeruginosaon isolated guinea pig tracheas to determine the antiasthma effects.Methods:The spasmolytic activity ofC. aeruginosaextracts was tested in separated organs of guinea pig trachea. Guinea pig was sacrificed and its trachea rings were suspended in L-shaped wire loops in organ baths containing the Krebs solution aerated with carbogen. Isometric contractions of tracheal rings were measured by the transducer coupled to the amplifier. The trachea rings were exposed to DMSO as negative control, aminophylline as positive control andC. aeruginosaextracts. The single concentration-relaxation curve was obtained in every preparation.Results:The result showed that the decrease of the spasmolytic activity in the guinea pig tracheal tone due toC. aeruginosaextract was significantly better (p=0.022) when compared to the negative control. Meanwhile, the EC50value of aminophylline (0.019 ± 0.05) was not significantly different (p=0.454) withC. aeruginosa(0.024 ± 0.05).Conclusion:It could be concluded thatC. aeruginosaextracts have the potency to be further developed as a new natural source of the antiasthma agents.

How to avoid severe coronary vasoconstriction in potassium induced cardioplegia

Objectives. The aim of this study was to investigate how high K+ concentrations can be safely used in cardioplegic solutions without causing severe coronary artery vasocontraction.Design. Twenty-four 50 kg pigs were used. The distal part of the left anterior descending coronary artery was cut into ring segments and transferred into organ baths with Krebs solution bubbled with 95% O2 and 5% CO2. K+ concentrations between 16 and 127 mM were used to induce vasocontractions at 37, 22, 15, and 8 °C. Mg2+ (0–20 mM) were used to attenuate K+ induced vasocontractions.Results. K+-Krebs solution 127 mM at 37 °C induced a strong, sustained vasocontraction defined as 100%. The contractions induced by 16, 23, 30 and 127 mM K+ were: 7.7, 38, 72 and 100% at 37 °C; 1.7, 7.4, 21 and 65% at 22 °C; 1, 6.6, 15 and 33% at 15 °C; 0.6, 2.1, 6 and 14% at 8 °C, respectively. Mg2+ reduced the K+-induced contraction at 37 °C in a concentration-dependent way and Mg2+ at 8 mM practically eliminated the risk for severe vasocontraction.Conclusions. Hypothermia (8 °C) abolishes coronary contraction induced by K+-cardioplegic solutions. In normothermic cardioplegia 8 mM Mg2+ prevents vasoconstriction.

Effects of Injections of Nanostructured Titanium Dioxide into the Rat Medullary Nuclei Involved in Cardiovascular Control

Effects of injections of titanium dioxide nanoparticles into the medullary nuclei involved in cardiovascular control (nuclei reticularis paramedianus, reticularis lateralis, and ambiguus) on the blood pressure and heart rate were examined in rats. The nanoparticles (TiO2, PlasmaChem GmbH, D-12489 Berlin, Germany) were used in the form of a nanopowder (mixture of rutile and anatase). The average particle size measured using a scanning electron microscope was 21 ± 5 nm, the specific surface area of the particles was 50 ± 10 m2/g, the purity exceeded 99.5%, the Al2O3 content was below 0.3 mass %, and the SiO2 content did not exceed 0.2%. The nanoparticles were dispersed in Krebs solution (pH 7.4) using ultrasonic cavitation processing for 2 min at 37 kHz. The zeta potential of TiO2 suspension was – 7.93 mV. To provide the aggregate stability of aqueous suspensions of TiO2 nanoparticles, the latter were mechanically stirred. Injections of nanosized TiO2 in the mentioned medullary nuclei induced noticeable changes in the blood pressure, which depended on the concentration and the site (nucleus) of injection. Changes in the heart rate were insignificant in all cases.

Determining the effect of long-term dexamethasone and prednisolone treatment on sugammadex

Background/aim: We aimed to investigate the effect of long-term use of dexamethasone and prednisolone on the reversal effect of sugammadex. Materials and methods: TTwenty-four male Wistar albino rats were divided into three groups. Dexamethasone (600 µg/kg) was given to group D, prednisolone (10 mg/kg) was given to group P, and an equivalent volume of saline per day was administered intraperitoneally to group S for 14 days, respectively. The left hemidiaphragm with attached phrenic nerve was maintained in Krebs solution. Sugammadex (30 µmol/L) was applied while rocuronium (10 µmol/L) was present in an organ bath and a single twitch was obtained. The right hemidiaphragm was used for both adult ( ε-subunit) and fetal nicotinic acetylcholine receptor (AChR) ( ε-subunit) determination using polymerase chain reaction. Results: All animals lost weight, except group S. The mean baseline single-twitch tension was lower in both group D (14.4 ± 1.7 g) and group P (12.68 ± 0.05 g) than group S (16.8 ± 0.5 g) (P < 0.001). When sugammadex was added to the organ bath while rocuronium was present, the single twitch was measured to be lower in both group D (11.7 ± 0.7 g) and group P (11.5 ± 0.78 g) than group S (16.5 ± 0.24 g) (P < 0.001). Ɣ-AChR expression was higher in both dexamethasone and prednisolone than in saline. Conclusion: Long-term medication with dexamethasone and prednisolone caused muscle weakness, resistance to neuromuscular blockers, and upregulation of immature Ɣ-AChR and reduced the neuromuscular reversal effect of sugammadex.

Stress-strain analysis of duodenal contractility in response to flow and ramp distension in rabbits fed low-fiber diet.

Previously we demonstrated that low-fiber diet in rabbits affects the passive mechanomorphological properties in the small intestine, resulting in reduced intestinal wall thickness and collagen content, as well as intestinal wall softening. The aim of the present study was to evaluate the contractility in rabbits on long-term low-fiber diet and specifically to compare the contraction threshold, the frequency, and the amplitude of flow-induced and distension-induced contractions in the duodenum between rabbits on normal diet and on long-term low-fiber diet. Ten rabbits were fed a low-fiber diet for 5months (Intervention group), and five rabbits were fed normal diet (Control group). The duodenal segments were used for determination of mechanical parameters for analyses of contractility. The duodenal experiments were carried out in organ baths containing physiological Krebs solution. Pressure and diameter changes induced by contractions in response to flow and ramp distension were measured. The frequencies and amplitude of contractions were analyzed. Distension-induced contraction thresholds and maximum contraction amplitude of flow-induced contractions were calculated in terms of mechanical stress and strain. Multiple linear regression analyses were applied to study dependencies between contractility parameters and wall thickness, wall area, and muscle layer thickness. During distension, the pressure, stress, and strain thresholds for induction of phasic contraction were biggest in the Intervention Group (P<0.05). In addition, the contraction frequencies during flow-induced contraction were highest in the Intervention Group (P<0.05), whereas the maximum contraction amplitudes in terms of pressure, diameter, stress, and strain were lowest in the Intervention Group (P<0.05). The contraction thresholds and contraction frequencies were negatively associated with the wall thickness, wall area, and muscle layer thickness, whereas maximum contraction amplitudes were positively associated with the wall thickness, wall area, and muscle layer thickness. Duodenal contractility in rabbits fed with long-term low-fiber diet exhibited low contraction amplitudes and high contraction thresholds and frequencies. The changes were associated with the low-fiber diet-induced histomorphological remodeling. Studies on detailed structural and functional diet-induced changes in smooth muscle and intestinal nerves are needed for better understanding the remodeling mechanisms.

Role of different nicotinic acetylcholine receptor subtypes in skeletal muscles in sepsis-related nerve-muscle dysfunction in septic rats: neuroelectrophysiology

Objective To evaluate the role of different nicotinic acetylcholine receptor (nAChR) subtypes in skeletal muscles in sepsis-related nerve-muscle dysfunction in septic rats by neuroelectrophysiology. Methods SPF adult male Sprague-Dawley rats, weighing 200-220 g, aged 2-3 months, were studied.Sepsis was induced by cecal ligation and puncture in chloral hydrate-anesthetized rats.Twelve portions of sciatic nerve-tibialis anterior muscles taken at day 3 after successful establishment of the sepsis model served as group S. Another 12 portions of sciatic nerve-tibialis anterior muscles of normal rats served as group C. The endplate potential (EPP) was monitored by intracellular microelectrode recording.Six portions of nerve-muscle samples were incubated with the adult nAChR (e-nAChR) specific antagonist waglerin-1 at a concentration of 1 μmol/L, the fetal nAChR (γ-nAChR) specific antagonist αA-OIVA at a concentration of 250 nmol/L and neuronal nAChR (α7-nAChR) antagonist methyllycaconitine (MLA) at a final concentration of 6 nmol/L.The amplitude of EPP was recorded at 15 min of incubation, and the difference in the amplitude of EPP (ΔEPP) and changing rate of EPP (ΔEPP%) were calculated.Another 6 portions of samples were incubated for 15 min with the Kreb mixture of waglerin-1 and 250 nmol/L αA-OIVA at a final concentration of 1 μmol/L, the Kreb mixture of waglerin-1 and 6 nmol/L MLA at a final concentration of 1 μmol/L, and the Kreb mixture of αA-OIVA and 6 nmol/L MLA at a final concentration of 250 nmol/L, and then the amplitude of EPP was recorded.Washout was performed with antagonist free Kreb solution for 15 min between treatments with different blockers. Results Compared with group C, ΔEPP and ΔEPP% were significantly decreased after antagonizing e-nAChR, ΔEPP% was decreased after antagonizing γ-nAChR, ΔEPP and ΔEPP% were increased after antagonizing α7-nAChR, the amplitude of EPP was increased after antagonizing e-nAChR+ γ-nAChR and e-nAChR+ α7-nAChR, and the amplitude of EPP was decreased after antagonizing γ-nAChR+ α7-nAChR (P<0.05). ΔEPP and ΔEPP% were significantly higher after antagonizing α7-nAChR than after antagonizing γ-nAChR in group S (P<0.05). The EPP was significantly lower after antagonizing e-nAChR+ α7-nAChR than after antagonizing e-nAChR+ γ-nAChR (P<0.05). Conclusion Weakened function of e-nAChR and enhanced function of α7-nAChR in the membrane of the end-plate in skeletal muscle are the main reasons for sepsis-related nerve-muscle dysfunction in septic rats, and the role is comparable between them. Key words: Sepsis; Neuromuscular diseases; Receptors, cholinergic

Skeletal muscle secretion of IL-6 is muscle type specific: Ex vivo evidence

Emerging evidence indicates that skeletal muscle possesses endocrine function to secret myokines. Interleukin 6 (IL-6) is a well-characterized myokine that is involved in regulation of metabolism and muscle function. Metabolism type and contractile dynamics vary in different muscle types. It is not clear, however, if IL-6 secretion differs in different muscle types. In this study, we first established an ex vivo approach to test the inducible muscle secretion. Freshly isolated muscles were incubated in Krebs solution at 37 °C with oxygen supply. Secreted IL-6 in the incubation media was measure using Western blot and ELISA assay. We first confirmed that the IL-6 release was inducible by treating the incubated muscle with a cytokine stimulant. We demonstrated that physiological temperature (37 °C) and O2 supply were essential for the induction of IL-6 release from the incubated muscle, suggesting it is a controlled secretion rather than a spontaneous leak. Using this approach, we found that IL-6 release was only inducible from soleus muscle but not EDL muscle. We further showed that IL-6 protein level was higher in slow oxidative muscle fibers. Moreover, we showed that EDL, although lacks of IL-6 release, surely has inducible secretory function that had different secretory pattern from soleus.

Potential Limitation for the use of Cyclosporine A as a Cardioprotective Agent during Donor Heart Retrieval and Storage

Background The success of heart transplantation has allowed the consideration of older and sicker recipients for transplant but has necessitated consideration of hearts from “extended criteria” donors for transplant. We have successfully repurposed glyceryl trinitrate and erythropoietin for clinical use as additives to cardioplegic or protective cold flush solutions for routine clinical retrieval of marginal hearts from donors after both brain death or circulatory death (1, 2). Introduction of normothemic perfusion of the heart during transportation as an alternative to static cold storage has presented an opportunity to bolster the initial protective flush and the normothermic perfusate with extra cardioprotective agents. One such potential candidate is Cyclosporine A (CsA). It was shown to decrease cardiac ischemia reperfusion injury in pre-clinical models by minimizing mitochondrial permeability transition pore opening. The present study investigates the role of timing and duration of cardiac exposure to CsA during retrieval and storage on functional recovery and mechanisms of CsA action in a working rat heart model of donor heart preservation. Methods After measurement of baseline function, hearts were arrested and stored for 6h at 4°C in either Celsior® alone or Celsior®+CsA (0.2μM), then reperfused for 45min in Krebs solution, when functional recovery was assessed. Two additional groups of Celsior®-alone stored hearts were exposed to 0.2μM CsA for the initial 15min (non-working period) or the full 45min period of reperfusion. Coronary effluent was collected pre- and post-storage for assessment of LDH release. Left ventricular free wall was snap-frozen after completion of each study for immunoblotting. Results Presence of CsA during storage or the first 15min reperfusion significantly improved functional recovery and significantly increased phospho-AMPKThr172 and phospho-ULK-1Ser757. Hearts exposed to CsA for 45min post-reperfusion recovered poorly with no phospho-AMPK increase, decreased mitochondrial cyctochrome c content and increased LDH release. Conclusions Inclusion of CsA at cardioplegia is cardioprotective. No extra benefit was gained by addition of CsA during the initial 15min reperfusion period. Presence of CsA for the full 45min reperfusion was toxic to the heart. Protective effects of CsA appear to be driven by activation of AMP associated protein kinase.

A MINIMAL ROLE FOR ENDOTHELIAL NITRIC OXIDE IN VASOCONSTRICTOR AND VASODILATOR RESPONSES OF GUINEA-PIG AORTA

Objective: Acetylcholine (Ach) dilates rat isolated aorta by releasing nitric oxide (NO) from the vascular endothelium. The vasoconstrictors, U46619 (thromboxane mimetic), phenylephrine (α-adrenoceptor agonist) and β-phenylethylamine (PEA) (trace amine associated receptor (TAAR) agonist), also release NO causing opposing vasodilatation. The roles of NO and endothelium in these vasoconstrictor responses were examined in guinea-pig aorta, along with the status of the endothelium. Design and method: Isometric contractions of guinea-pig aortic rings immersed in Kreb's solution gassed with 5% CO2 in O2 at 37 ± 0.5oC were recorded. Aortae were either intact or denuded of endothelium by rubbing. Cumulative concentration-response curves (CRC) for acetylcholine chloride, β-phenylethylamine hydrochloride (PEA) or (-)-phenylephrine HCl were obtained. Contractions were expressed as a percentage of the contraction to KCl (60 mM). Responses were compared by Student's paired t-test, P less than 0.05 indicating significance. Results: Contractions to phenylephrine and PEA were not significantly different in endothelium intact and denuded aortae. The maxima for phenylephrine were 80.4 ± 3.0 (n = 11) and 77.1 ± 8.1% (n = 6), respectively and the maxima for PEA were 71.7 ± 3.6 (n = 22) and 60.2 ± 3.4% (n = 17), respectively. Contractions to U46619 (1 μM) were also not significantly different in intact (1.32 ± 0.14 g n = 4) and denuded tissues (1.24 ± 0.38 (n = 12). We tested whether endothelium was present in intact tissues by adding Ach to U46619-pre-contracted aortae (1 or 0.5 μM). Of 16 intact tissues, only 6 responded with small falls in tension (0.07 ± 0.02 g), representing only 7.11 ± 3.04% of the contraction to U46619 (1.33 ± 0.27 g). Sodium nitroprusside, however, relaxed U46619-precontracted (1 μM) aorta reaching a maximum of 97.3 ± 5.1% of the U46619 contraction (1.58 ± 0.21 g, n = 4). No denuded preparations responded to Ach. Ach in non-contracted intact aortae produced small dose-related contractions. After washout (x2), tissues were raised from the bath and the endothelium removed before returning to the bath for a second CRC, which was potentiated, the maximum increasing from 25.1 ± 10.5% to 48.9 ± 21.5% (n = 4). Phenylephrine contractions also increased after rubbing to remove endothelium. Conclusions: Intact guinea-pig aortae displayed poor relaxations to Ach suggesting a minimal role of endothelial muscarinic responses. In uncontracted aortae, Ach caused constriction which was potentiated when repeated after endothelium removal, indicating a minor opposing endothelium-dependent vasodilatation.

The effect of magnesium sulfate concentration on the effective concentration of rocuronium, and sugammadex-mediated reversal, in isolated left phrenic nerve hemi-diaphragm preparations from the rat.

BackgroundPerioperative magnesium sulfate (MgSO4) is used for analgesic, anti-arrhythmic, and obstetric purposes. The effects of MgSO4 on the neuromuscular blockade (NMB) induced by rocuronium, and the sugammadex reversal thereof, have not been clearly quantified. We investigated the effect of various MgSO4 concentrations on the NMB by rocuronium, and sugammadex reversal, in isolated left phrenic nerve hemi-diaphragm (PNHD) preparations from the rat.MethodsRat PNHD preparations were randomly allocated to one of four groups varying in terms of MgSO4 concentration (1, 2, 3, and 4 mM, each n = 10, in Krebs solution). The train-of-four (TOF) and twitch height responses were recorded mechanomyographically. The preparations were treated with incrementally increasing doses of rocuronium and each group’s effective concentration (EC)50, EC90, and EC95 of rocuronium were calculated via nonlinear regression. Then, sugammadex was administered in doses equimolar to rocuronium. The recovery index, time to T1 height > 95% of control, and the time to a TOF ratio > 0.9 after sugammadex administration were measured.ResultsThe EC50, EC90, and EC95 of rocuronium fell significantly as the magnesium level increased. The EC50, EC90, and EC95 of rocuronium did not differ between the 3 and 4 mM groups. The recovery index, time to T1 height > 95% of control, and time to a TOF ratio > 0.9 after sugammadex administration did not differ among the four groups.ConclusionsIncreases in the magnesium concentration in rat PNHD preparations proportionally enhanced the NMB induced by rocuronium but did not affect reversal by equimolar amounts of sugammadex.

The effects of proton pump inhibitor on hepatic vascular responsiveness and hemodynamics in cirrhotic rats

BackgroundLiver cirrhosis is associated with increased intrahepatic resistance due to hepatic fibrosis and exaggerated vasoconstriction. Recent studies have indicated that proton pump inhibitors (PPIs), in addition to acid suppression, modulate vasoactive substances and vasoresponsiveness. PPIs are frequently prescribed in patients with cirrhosis due to a higher prevalence of peptic ulcers, however other impacts are unknown. MethodsLiver cirrhosis was induced in Sprague–Dawley rats with common bile duct ligation (BDL). On the 29th day after BDL and after hemodynamic measurements, the intrahepatic vascular responsiveness to high concentrations of endothelin-1 (ET-1) was evaluated after preincubation with (1) Krebs solution (vehicle), (2) esomeprazole (30 μM), or (3) esomeprazole plus Nω-nitro l-arginine (NNA, a non-selective NO synthase (NOS) inhibitor, 10−4 M). After perfusion, the hepatic protein expressions of endothelial NOS (eNOS), inducible NOS (iNOS), cyclooxygenase (COX)-1, COX-2, endothelin-1, DDAH-1 (dimethylarginine dimethylaminohydrolase-1, ADMA inhibitor), DDAH-2, ADMA (asymmetrical dimethyl arginine, NOS inhibitor) were evaluated. In the chronic model, the BDL rats received (1) vehicle; or (2) esomeprazole (3.6 mg/kg/day, oral gavage) from the 1st to 28th day after BDL. On the 29th day and after hemodynamic measurements, plasma liver biochemistry and liver fibrosis were evaluated. ResultsEsomeprazole did not affect hepatic ET-1 vasoresponsiveness. The hepatic protein expressions of the aforementioned factors were not significantly different among the groups. There were no significant differences in hemodynamics, liver biochemistry and hepatic fibrosis after chronic esomeprazole administration. ConclusionPPIs did not affect hepatic vasoresponsiveness or the release of vasoactive substances. Furthermore, they did not influence hemodynamics, liver biochemistry or severity of hepatic fibrosis in the cirrhotic rats.

Senescent Skeletal Muscle Produces a Distinct Cytokine Secretory Profile in Response to Endotoxin Exposure in Vitro

Skeletal muscles are thought to contribute to both pathogen detection and cytokine secretion as a part of the innate immune response. Skeletal muscle fibers contain toll like receptors (TLRs) that bind a wide array of bacterial, viral, and fungal ligands, inducing signal transduction and subsequent cytokine production. Aging populations have significantly greater mortality annually from influenza, incidence of bacterial and fungal infections, and diminished vaccine efficacy. In senescent mice, macrophage TLR expression and cytokine secretion are known to decline in some tissues. It is unknown if skeletal muscle also exhibits age associated changes in innate immune contribution. The objective of this experiment was to determine if there are alterations in cytokine secretory response of aged skeletal muscle to lipopolysaccharide (LPS), a potent TLR2 and TLR4 ligand.Matched solei muscles from 12–15‐week‐old male C57BL/6 mice (n=8; “young”) and 16‐month‐old (n=10; “old”) were excised in oxygenated (95%O2/5%CO2) Krebs solution and placed under 1 g of tension in 2 ml baths at 35°C. After a 30‐min acclimation period (T0), the buffer was changed and muscles were exposed to 1 μg/ml LPS. Bath samples at T0, 1 hr (T1) and 2 hr (T2) were taken, combined with a protease inhibitor cocktail and frozen at −80°C prior to Luminex multiplex analysis.In conclusion, solei from old mice generally displayed a diminished cytokine secretory response, with particularly large decrements in IL‐12p70, IL‐6, MCP‐1, KC, and G‐CSF compared to young controls (see Table 1). Interestingly, solei from old mice displayed relatively large increases in IL‐1α, with smaller, yet also significant increases in IL‐10 and TNFα. The drastically lowered IL‐6 secretion in older mice may diminish the normal inhibition on IL‐1α and subsequent TNFα production. The small rise in IL‐10 in the elder mice may serve as a compensatory mechanism to lower IL‐1α due to diminished IL‐6 response. It may also contribute towards the severely lowered KC production, as is seen with the inhibition of human KC homologue IL‐8 by elevated IL‐10. The combination of radically lower IL‐6 and KC secretion, and increased IL‐1α and TNFα may produce an environment favoring excessive inflammatory, yet reduced chemotactic responses to pathogen exposure in older mice.Support or Funding InformationSupported by NIGMS R01GM118895‐01 and the BK and Betty Stevens Endowment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fenugreek (Trigonella Foenum-Graecum) Seed Flour and Diosgenin Preserve Endothelium-Dependent Arterial Relaxation in a Rat Model of Early-Stage Metabolic Syndrome.

Fenugreek is a common herb possessing several bioactive components including diosgenin. Here, dietary fenugreek seed flour and diosgenin were evaluated on a model of endothelium-dependent vasorelaxation by abdominal aortas isolated from rats receiving high-fat, high-sugar diet (HFHSD). 60 male Wistar rats were randomized into six groups: (i) negative control getting conventional rat feed regimen; (ii) positive control receiving HFHSD; (iii) a test group fed 2 g/kg bw/day fenugreek seed flour (containing 10 mg/kg bw/day diosgenin) + HFHSD; (iv) three test groups fed 1, 10 and 50 mg/kg bw/day diosgenin + HFHSD. Alimentary treatments were carried out for six weeks. The abdominal aortas were isolated, and 2 mm wide rings were sectioned off and mounted at a resting tension of 10 mN in organ baths containing Krebs solution (36 °C) exposed to 95% O2 and 5% CO2. After 60-min incubation, a norepinephrine concentration-response (E/c) curve was generated to determine their half-maximal effective concentration (EC50) value. After 60-min wash-out, a pre-contraction with norepinephrine EC50 was made, followed by an acetylcholine E/c curve. Plasma glutathione levels, glutathione-handling enzyme activities and blood antioxidant capacities were also determined. HFHSD significantly decreased the dilatory response to acetylcholine and increased plasma glutathione levels and these effects were significantly reversed by fenugreek seed flour, 10 and 50 mg/kg bw/day diosgenin. Both fenugreek and diosgenin treatments prevent HFHSD-induced endothelial dysfunction and redox changes. As fenugreek treatment was more effective at lower acetylcholine concentrations than diosgenin treatments, components of fenugreek other than diosgenin may contribute to the beneficial effects of dietary fenugreek seed flour.