Krebs-Ringer Bicarbonate Solution Research Articles

Vasorelaxant effect of monoterpene carvacrol on isolated human umbilical artery.

Carvacrol (CRV) is the main compound of essential oils extracted primarily from Thymus and Origanum species. Its various biological activities were confirmed: antioxidant, anti-inflammatory, antibacterial, antifungal, anti-tumour, antinematodal, and vasorelaxant action. Although vasodilation mediated by CRV was previously described, the exact mechanism of its action has not yet been established. Hence, the aim of this study was to investigate CRV vasoactivity on human umbilical arteries (HUA) and the different pathways involved in its mechanism of action using the tissue bath methodology. CRV caused a significant decrease in vascular tension of 5-HT-pre-contracted umbilical arteries, with EC50 of 442.13±33.8µmol/L (mean±standard error of the mean-SEM). At 300µmol/L, CRV shifted downward the 5-HT concentration-response curve with a statistical significance of p<0.001 obtained for the four highest concentrations. At a concentration of 1mmol/L, CRV completely abolished BaCl2-induced contraction in Ca2+-free Krebs-Ringer bicarbonate solution and the BAY K 8644-induced contraction in Krebs-Ringer bicarbonate solution (p<0.001). Isopentenyl pyrophosphate, the antagonist of TRPV3 channel, was able to decrease the efficacy of CRV (p<0.001). The blocking of L-type Ca2+ channels on smooth muscle cells is the most probable mechanism of CRV-induced vasorelaxation. However, the role of TRPV3 channels in CRV-induced vasodilation of HUA cannot be excluded either.

The effects of nitrofurantoin on rat urinary bladder contraction

Nitrofurantoin is a drug used in the antibacterial treatment of urinary tract infections (UTIs) for over 60 years. The aim of our study was to investigate the possible effects of nitrofurantoin, which is commonly used as a urinary tract antiseptic, on bladder contractions in male rats. Bladder tissues obtained from male Sprague-Dawley rats were used in the study (n=24). After decapitation, bladder tissues were suspended in an isolated organ bath of 5 ml containing a Krebs-Ringer bicarbonate solution by applying a tension of 1.5 grams. Nitrofurantoin was administered to three groups at doses of 50, 500 and 1000 μM, respectively. The area, peak-to-peak (p-p) and frequency values of bladder contractions were analyzed before and after administration of nitrofurantoin. The data obtained from the analysis were evaluated using the Paired T-Test in the IBM SPSS Statistics Software. Nitrofurantoin was observed to have an inhibitory effect on bladder contractions at all doses. The decrease in the area and peak-to-peak values was statistically significant at all doses (P

Clopidogrel inhibits acetylcholine-induced contractions of urinary bladder in rat

Aim: Clopidogrel as thrombocyte Adenosine Diphosphate (ADP) receptor antagonist is used especially in peripheric artery diseases by lengthening hemorrhage time, disrupting thrombocyte aggregation and decreasing blood viscosity. It shows its antagonist effects through glycoprotein (Gp) IIb/IIIa complex ADP by preventing its activation. Although the usage area and activity is in the vascular system, there are no adequate studies showing the activity of Clopidogrel on smooth muscle contraction-relaxation mechanism. This study was conducted to investigate the effects of Clopidogrel on bladder contraction-relaxation mechanism.Material and Methods: In the present study, the bladder tissues taken from Wistar-Albino (n=7) intact female rats were used. After the decapitation, the longitudinal bladder tissues that were received 1-mm-thick, 8-mm length, and 2-mm-width, were hung in the 5-ml isolated organ bath that had Krebs-Ringer bicarbonate solution by applying 1.5 gr strain. After the bladder contractions were induced with 10 μM dose Acetylcholine (Ach), Clopidogrel was applied as two doses 0.1μM and 10μM in a noncumulative manner. The peak-to-peak (p-p) values and the values below the curve before and after the Clopidogrel application in the contraction induced with Ach were normalized as % change. The statistical analyses of the data were made in the SPSS 22.0 program by applying Paired T-Test. The p

Investigation the relaxant effects of proton pump ınhibitors and their relaxation mechanism on sheep sphincter of Oddi.

Using a relaxant agent before an endoscopic retrograde cholangiopancreatography (ERCP) might reduce complications. We aimed to investigate the relaxant effects of proton pump inhibitors (PPIs) on sheep sphincter of Oddi (SO) and the mechanisms that might take part in this relaxant effect. The sheep SO was mounted in an organ bath filled with Krebs-Ringer bicarbonate solution under 1.5 g tension and the relaxant effects of PPIs were evaluated in the tissues precontracted by carbachol (10-6 mol/l). The relaxant responses to the PPIs were tested in the presence of various blockers to enlighten the underlying mechanism by the PPIs. The PPIs exerted relaxant responses in a concentration-dependent manner in the sheep SO (P < 0.05). Esomeprazole produced the strongest relaxation. The administration of atropine, indomethacin, L-NAME, methylene blue, clotrimazole, glibenclamide, and 4-aminopyridine into the organ baths did not change the relaxations induced by PPIs in vitro (P> 0.05). On the other hand, Ca+2-activated potassium channel blocker tetraethylammonium (TEA) reduced the relaxation responses created by PPIs (P < 0.05). The present study suggests that PPIs create relaxation on SO partially via Ca+2-activated potassium channels. PPIs, especially esomeprazole, may be beneficial during the ERCP procedure. Further clinical studies are needed to confirm our results.

Clarification of serotonin-induced effects in peripheral artery disease observed through the femoral artery response in models of diabetes and vascular occlusion: The role of calcium ions.

Recent findings have demonstrated that serotonin is an important participant in the development and progression of peripheral artery diseases. Taking this into consideration, the goals of this study were to investigate the effects of serotonin on isolated Wistar rat femoral arteries in both healthy and diabetic animals, with and without artery occlusion, with a particular focus on determining the role of calcium in this process. Contraction experiments with serotonin on intact and denuded femoral artery rings, in the presence or absence of nifedipine and ouabain (both separately, or in combination), as well as Ca2+ -free Krebs-Ringer bicarbonate solution were performed. The serotonin-induced results were concentration dependent, but only in healthy animals. The endothelium-dependent contraction of the femoral artery was assessed. In healthy animals, the endothelium-reliant part of contraction was dependent on the extracellular calcium, while the smooth muscle-related part was instead dependent on the intracellular calcium. In diabetic animals, both nifedipine and ouabain influenced serotonin-induced vascular effects by blocking intracellular calcium pathways. However, this was diminished after the simultaneous administration of both blockers.

Vasorelaxing Effect of Resveratrol on Bovine Retinal Arteries.

Resveratrol is a red wine polyphenol that causes vasorelaxation, which could be of interest in the treatment or prevention of eye diseases with an impaired blood flow. In this study, the vasorelaxant capacity of resveratrol (cis and trans) on bovine retinal arteries, its vasorelaxing mechanism, and its influence on the relaxation induced by the retinal relaxing factor (RRF) were examined. Isolated bovine retinal arteries were mounted into wire myographs for isometric tension measurements. Concentration-response curves of cis- and trans-resveratrol and concentration-response curves of resveratrol in the absence or presence of the endothelium or different inhibitors were constructed. Relaxations elicited by the RRF with and without resveratrol incubation were also compared. Both resveratrol isomers caused a similar strong concentration-dependent relaxation. Removal of the endothelium or blocking endothelium-dependent pathways did not change the relaxation. Also, K+ channel blockers did not reduce the relaxation, except the 120 mM K+ Krebs Ringer bicarbonate solution. Phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate blocked the relaxation partially and so did the inhibition of heme oxygenase-1. Blocking adenylyl cyclase, AMP-activated protein kinase, estrogen receptors, sirtuin 1, or sarco/endoplasmic reticulum Ca2+ ATPase did not have an effect. The relaxation caused by the RRF was not altered by resveratrol incubation. Cis- and trans-resveratrol relax bovine retinal arteries similarly and concentration dependently. The main relaxation mechanism remains unclear, but K+ channels, carbon monoxide, and the myosin phosphatase pathway may be involved. Resveratrol does not have an influence on the RRF.

The Early History of the TYH Medium forin vitroFertilization of Mouse Ova

Abstract: The TYH medium was first reported as a medium for in vitro fertilization (IVF) of mouse eggs with epididymal spermatozoa, by Toyoda, Yokoyama and Hosi, in 1971. It was a modified Krebs-Ringer bicarbonate solution, supplemented with glucose, Na-pyruvate, antibiotics and bovine serum albumin. In TYH medium, almost all eggs are fertilized within 1 h, if the spermatozoa are pre-incubated for 2 h before insemination, while the sperm penetration is delayed for approximately 1 h when fresh epididymal spermatozoa are used. These findings showed that sperm capacitation can be induced in a chemically defined medium without requiring the female reproductive tract. Although the medium was not specifically named in the original paper, it was later called “TYH” after the initials of the three authors of the original paper. The IVF method using TYH medium is widely used due to its high reproducibility. In this mini-review, we describe the early efforts to develop the TYH medium and briefly discuss the related ...

High-Dose Radiation-Induced Changes in Murine Small Intestinal Motility: Are the Changes in the Interstitial Cells of Cajal or in the Enteric Nervous System?

Murine small intestinal motility consists of phasic contraction from interstitial cells of Cajal (ICC) and migrating motor complexes (MMCs) from the enteric nervous system. The number of ICC is reduced in various gastrointestinal disorders, and this effect can be reversed once the disorder is resolved through cellular and tissue remodelling. Exposure to high-dose radiation can induce inflammation and alter intestinal motility. In this study, we investigated the changes in the small intestinal motility of 8- to 10-week-old male C3H/HeN mice after high-dose (13 Gy) irradiation. The aim of this study was to determine whether those changes are caused by changes in the ICC or enteric nervous system. After irradiation, the small intestine was dissected and stored in oxygenated Krebs-Ringer bicarbonate solution. The tension of contractions and intracellular membrane potentials were recorded at day 0, 1, 3 and 5 after irradiation and compared with those of sham-irradiated mice. Histological evaluation was performed by immunohistochemistry and apoptosis was evaluated. Quantitative real-time polymerase chain reaction (qPCR) for c-kit mRNA was also performed. Phasic contractions were not changed at day 0, 1, 3 and 5 after irradiation and did not significantly differ from those in the control mice. Slow waves were also sustained after irradiation. However, the frequency of migrating motor complexes (MMCs) was significantly higher at day 0 and 1 after exposure and the amplitude and area under the curve were significantly lower at day 3 after exposure compared with control mice. MMCs were recovered at day 5 with no difference from those of the control mice. ICC were detected after irradiation by immunohistochemistry for c-kit, and c-kit mRNA levels did not differ between sham-irradiated and irradiated mice. Histological evaluation showed that the most severe inflammation was detected at day 3 after irradiation, and apoptosis was detected only in the mucosa. Acetylcholine increased the contractility after irradiation, and tetrodotoxin decreased the number of MMCs in sham-irradiated and irradiated mice. N(w)-oxide-l-arginine (L-NA) increased the number of MMCs. MMCs were recovered after L-NA treatment at day 3 after irradiation. Sodium nitroprusside decreased the MMCs in sham-irradiated and irradiated mice. Exposure to high-dose radiation did not alter phasic contractions and slow waves in the small intestine of mice, which suggests that ICC and their functions may be sustained after high-dose irradiation. Mucosal inflammation was severe after irradiation and there were some changes in MMCs related to the enteric nervous system.

A Sensitive Medium-Throughput Method to Predict Intestinal Absorption in Humans Using Rat Intestinal Tissue Segments

A range of in vitro, ex vivo, and in vivo approaches are currently used for drug development. Highly predictive human intestinal absorption models remain lagging behind the times because of numerous variables concerning permeability through gastrointestinal tract in humans. However, there is a clear need for a drug permeability model early in the drug development process that can balance the requirements for high throughput and effective predictive potential. The present study developed a medium throughput screening Snapwell (MTS-Snapwell) ex vivo model to provide an alternative method to classify drug permeability. Rat small intestine tissue segments were mounted in commercial Snapwell™ inserts. Unidirectional drug transport (A-B) was measured by collecting samples at different time points. Viability of intestinal tissue segments was measured by examining transepithelial electric resistance (TEER) and phenol red and caffeine transport. As a result, the apparent permeability (Papp; ×10(-6) cm/s) was determined for atenolol (10.7 ± 1.2), caffeine (17.6 ± 3.1), cimetidine (6.9 ± 0.1), metoprolol (12.6 ± 0.7), theophylline (15.3 ± 1.6) and, ranitidine (3.8 ± 0.4). All drugs were classified in high/low permeability according to Biopharmaceutics Classification System showing high correlation with human data (r = 0.89). These findings showed a high correlation with human data (r = 0.89), suggesting that this model has potential predictive capacity for paracellular and transcellular passively absorbed molecules.

Contractile function of smooth muscle retained after overnight storage.

The functional responses of different overnight-stored in vitro tissues are not clearly described in any animal model. The influence of overnight storage in an animal model may vary between tissue types. We employed Sprague-Dawley rat as our animal model and investigated the functional changes of rat aorta, trachea, bronchus and bladder that were used (i) immediately after surgical removal (denoted as fresh) and (ii) after storage in aerated (95% O2, 5% CO2) Krebs-Ringer bicarbonate solution at 4 °C for 24 h (denoted as stored). The aorta ring was pre-contracted with phenylephrine, and the functional response of the tissue was investigated using isoprenaline, forskolin and carbachol. Carbachol was also used to increase the tone in trachea, bronchus rings and bladder strips. A clear reduced function of endothelium, with a minor if any effect in the smooth muscle function in rat aorta was observed after overnight storage. The contractile response of overnight-stored rat airway (trachea and bronchus) and bladder smooth muscles remained unchanged. Among all tested tissues, only bronchus showed a reduced response rate (only 40% responded) after storage. In vitro rat tissues that are stored in Krebs solution at 4 °C for 24 h can still be used to investigate smooth muscle responses, however, not endothelium-mediated responses for aorta. The influence of overnight storage on different tissues from an animal model (Sprague-Dawley rat in our study) also provides an insight in maximising the use of sacrificed animals.

The effects of eupatilin (stillen®) on motility of human lower gastrointestinal tracts.

Gastrointestinal motility consists of phasic slow-wave contractions and the migrating motor complex (MMC). Eupatilin (Stillen®) has been widely used to treat gastritis and peptic ulcers, and various cytokines and neuropeptides are thought to be involved, which can affect gastrointestinal motility. We performed a study to identify the effects of eupatilin on lower gastrointestinal motility with electromechanical recordings of smooth muscles in the human ileum and colon. Ileum and colon samples were obtained from patients undergoing bowel resection. The tissues were immediately stored in oxygenated Krebs-Ringer's bicarbonate solution, and conventional microelectrode recordings from muscle cells and tension recordings from muscle strips and ileal or colonic segments were performed. Eupatilin was perfused into the tissue chamber, and changes in membrane potentials and contractions were measured. Hyperpolarization of resting membrane potential (RMP) was observed after administration of eupatilin. The amplitude, AUC, and frequency of tension recordings from circular and longitudinal smooth muscle strips and bowel segments of the ileum and colon were significantly decreased after admission of eupatilin. Eupatilin elicited dose-dependent decreases during segmental tension recordings. In conclusion, eupatilin (Stillen®) showed inhibitory effects on the human ileum and colon. We propose that this drug may be useful for treating diseases that increase bowel motility, but further studies are necessary.

Role of calcium channels responsible for phenylephrine-induced contraction in rat aorta 3 days after acute myocardial infarction

BackgroundPhenylephrine (PE) produces tonic contraction through involvement of various calcium channels such as store-operated calcium channels (SOCCs) and voltage-operated calcium channels (VOCCs). However, the relative contribution of each calcium channel to PE-induced contraction has not been investigated in isolated rat aorta of early acute myocardial infarction (AMI).MethodsEndothelium-denuded rat aortic rings from rats 3 days after AMI or sham-operated (SHAM) rats were prepared in an organ chamber with Krebs-Ringer bicarbonate solution for isometric tension recording. We assessed the PE dose-response relationships in 2.5 mM calcium medium for both groups. The same procedure was repeated using rings pretreated with the SOCC inhibitor 2-aminoethoxydiphenyl borate, sarco/endoplasmic-reticulum calcium ATPase inhibitor thapsigargin (TG), diacyl glycerol lipase inhibitor RHC80267, and sodium-calcium exchanger inhibitor 3,4-dichlorobenzamil hydrochloride for 30 minutes before addition of calcium. When ongoing tonic contraction was sustained, dose-response curves to the VOCC inhibitor nifedipine were obtained to assess the relative contribution of each calcium channel under various conditions.ResultsThe effect of SOCC induction with TG pretreatment on PE-induced contraction was significantly lower in the AMI group compared to the SHAM group. In addition, there were significant decreases in the sensitivity and efficacy of the VOCC inhibitor nifedipine on PE-induced contraction in the AMI group.ConclusionsResults suggest that the change of vascular reactivity of PE in rat aorta 3 days after AMI is characterized by a decreased contribution of L-type VOCCs. The enhanced VOCC-independent calcium entry mechanisms after AMI can be mediated by enhanced capacitative calcium entry through the activation of SOCCs.

Acute hypoxia activates store-operated Ca(2+) entry and increases intracellular Ca(2+) concentration in rat distal pulmonary venous smooth muscle cells.

Exposure to acute hypoxia causes vasoconstriction in both pulmonary arteries (PA) and pulmonary veins (PV). The mechanisms on the arterial side have been studied extensively. However, bare attention has been paid to the venous side. To investigate if acute hypoxia caused the increase of intracellular Ca(2+) concentration ([Ca(2+)]i), and Ca(2+) influx through store-operated calcium channels (SOCC) in pulmonary venous smooth muscle cells (PVSMCs). Fluorescent microscopy and fura-2 were used to measure effects of 4% O2 on [Ca(2+)]i and store-operated Ca(2+) entry (SOCE) in isolated rat distal PVSMCs. In PVSMCs perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca(2+) stores in the sarcoplasmic reticulum (SR) and nifedipine to prevent Ca(2+) entry through L-type voltage-depended Ca(2+) channels (VDCC), hypoxia markedly enhanced both the increase in [Ca(2+)]i caused by restoration of extracellular [Ca(2+)] and the rate at which extracellular Mn(2+) quenched fura-2 fluorescence. Moreover, the increased [Ca(2+)]i in PVSMCs perfused with normal salt solution was completely blocked by SOCC antagonists SKF-96365 and NiCl2 at concentrations that SOCE >85% was inhibited but [Ca(2+)]i responses to 60 mM KCl were not altered. On the contrary, L-type VDCC antagonist nifedipine inhibited increase in [Ca(2+)]i to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca(2+)]i caused by hypoxia was completely abolished by perfusion with Ca(2+)-free KRBS. These results suggest that acute hypoxia enhances SOCE via activating SOCCs, leading to increased [Ca(2+)]i in distal PVSMCs.

Isolation and biochemical analysis of vesicles from taurohyodeoxycholic acid-infused isolated perfused rat livers

To isolate biliary lipid-carrying vesicles from isolated perfused rat livers after taurohyodeoxycholic acid (THDC) infusion. Biliary lipid vesicles have been implicated in hepatic disease and THDC was used since it increases biliary phospholipid secretion. Rat livers were isolated and perfused via the hepatic portal vein with THDC dissolved in Krebs Ringer Bicarbonate solution, pH 7.4, containing 1 mmol/L CaCl2, 5 mmol/L glucose, a physiological amino acid mixture, 1% bovine serum albumin and 20% (v/v) washed human erythrocytes at a rate of 2000 nmol/min for 2 h. The livers were then removed, homogenized and subjected to centrifugation, and the microsomal fraction was obtained and further centrifuged at 350000 g for 90 min to obtain subcellular fractions. These were analyzed for total phospholipid, cholesterol, protein and alkaline phosphodiesterase I (PDE). No significant changes were observed in the total phospholipid, cholesterol and protein contents of the gradient fractions obtained from the microsomal preparation. However, the majority of the gradient fractions (ρ= 1.05-1.07 g/mL and ρ = 1.95-1.23 g/mL) obtained from THDC-infused livers had significantly higher PDE activity compared to the control livers. The low density gradient fraction (ρ = 1.05-1.07 g/mL) which was envisaged to contain the putative vesicle population isolated from THDC-perfused livers had relatively small amounts of phospholipids and protein when compared to the relevant control fractions; however, they displayed an increase in cholesterol and PDE activity. The phospholipids were also isolated by thin layer chromatography and subjected to fractionation by high performance liquid chromatography; however, no differences were observed in the pattern of the fatty acid composition of the phospholipids isolated from THDC and control perfused livers. The density gradient fractions (ρ = 1.10-1.23 g/mL) displayed an increase in all the parameters measured from both control and THDC-infused livers. No significant changes in biliary lipids were observed in the fractions from THDC-infused livers; however, PDE activity was significantly increased compared to the control livers.

Evaluation of Caspase-1 Activation and IL-1β Production in A Kainic Acid Microdyalisis Brain Injury Model

Intracerebral infusion of kainic acid (KA) by a microdialysis probe induces a focal swelling in the brain-perfused area which promotes inflammation (Compan et al., 2012; Oprica et al., 2003). The microdialysis technique allows the local in vivo perfusion of KA and the simultaneous collection of inflammatory mediators, and other neuroactive substances, released in the injured brain. This protocol also allows the perfusion of different solutions in each cerebral hemisphere at the same time. By perfusing KA in isotonic solution of Krebs-Ringer Bicarbonate (KRB) (280-290 mOsm) in one hippocampus and KA in hypertonic KRB solution (1,400-1,500 mOsm) in the contralateral side, we can evaluate in vivo the efficiency of hypertonic solutions in preventing inflammation induced by swelling after KA infusion. Once the inflammatory response has been induced, it is possible to infuse through the microdialysis probe a biotinylated specific inhibitor of caspase-1 allowing the detection of the brain regions and cells involved in IL-1 production in response to the injury (Oprica et al., 2003).

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.

Elimination of Teicoplanin by Adsorption to the Filter Membrane during Haemodiafiltration: Screening Experiments for Linezolid, Teicoplanin and Vancomycin followed by in vitro Haemodiafiltration Models for Teicoplanin

Pharmaceutical agents directed against methicillin-resistant Staphylococcus aureus can be eliminated during haemodiafiltration, not only by diffusion and ultrafiltration, but also by adsorption onto haemofilters. The latter may be affected by the binding of agents to serum albumin. The present study therefore investigated the affinity of anti-methicillin-resistant Staphylococcus aureus agents (teicoplanin, linezolid, vancomycin) for haemofilters and the pharmacokinetic properties of teicoplanin during haemodiafiltration. Linezolid, teicoplanin and vancomycin were first screened for their in vitro affinity for three different kinds of filter membranes: polysulfone, polyacrylonitrile and polymethylmethacrylate. Only teicoplanin showed significant filter-binding activity. An in vitro haemodiafiltration circulation model was then developed that incorporated a one-litre beaker containing Krebs-Ringer's bicarbonate solution with/without human albumin (0 or 3 g/dl) as an artificial plasma. Teicoplanin (initial concentration 50 µg/ml, representing the maximum plasma concentration (Cmax) resulting from a typical clinical dosage) was circulated throughout the beaker. Teicoplanin concentrations in the 'plasma' and ultrafiltrate were determined by high performance liquid chromatography. In the screening experiment, teicoplanin was predominantly adsorbed onto polysulfone and polymethylmethacrylate membranes. Furthermore, teicoplanin was primarily eliminated by adsorption onto these filters during in vitro haemodiafiltration. Albumin significantly reduced both haemodiafiltration clearance and the adsorption-dependent elimination, although there were complex but significant interactions between albumin and the filter membrane. Elimination of teicoplanin in an in vitro haemodiafiltration model was largely due to adsorption onto polysulfone and polymethylmethacrylate haemofilters. Future clinical studies should likely be designed to evaluate present recommendations of teicoplanin dosages in patients on haemodiafiltration.

The effect of emetine on myocardial catecholamine metabolism.

Abstract The effects of emetine on the uptake and release of noradrenaline from the isolated, perfused, rat heart and on the in vivo myocardial retention of noradrenaline have been studied. Hearts were removed from animals under light ether anaesthesia, transferred to a modified Langendorff perfusing apparatus and perfused with Krebs-Ringer bicarbonate solution at a rate of 5 ml min−1. The effect of emetine on the uptake of noradrenaline was determined by perfusing hearts for a period of 6 min with perfusate containing various concentrations of emetine and 100 ng ml−1 of [14C]noradrenaline (14C-NA). After 6 min, the emetine-treated hearts contained less (±)−14C-NA than control hearts. The degree of reduction increased with increasing concentrations of emetine. The release of noradrenaline was determined after first perfusing hearts with 14C-NA followed by perfusion with solution containing emetine (0–01 mg ml−1) but free of labelled noradrenaline for periods of 10, 20 and 60 min. It was found that by 60 min emetine significantly decreased myocardial 14C-NA. In the intact animal myocardial concentrations of 14C-NA were reduced 90 min after intravenous 14C-NA, followed immediately by intraperitoneal emetine (1 mg kg−1). These results suggest that the cardiac arrhythmia and irritability seen after emetine treatment in man may be partly due to alterations in catecholamine metabolism.

The effect of pancuronium on myocardial contraction and catecholamine metabolism.

The effects of pancuronium bromide infusion on the uptake and release of [14C] noradrenaline (14C-NA) by the isolated, perfused rat heart and on the chronotropic and inotropic activity of the isolated heart were evaluated. Hearts were removed from animals under light ether anaesthesia, transferred to a modified Langendorff perfusing apparatus and perfused with Krebs-Ringer bicarbonate solution at a rate of 5 ml min-1. The effect of pancuronium on the uptake of noradrenaline was determined by perfusing hearts for 5 min with perfusate containing various concentrations of pancuronium and 200 ng ml-1 of 14C-NA. After 5 min pancuronium-treated hearts contained less 14C-NA. The degree of reduced uptake increased with increasing concentrations of pancuronium. In addition, the combination of pancuronium perfusion and electrical stimulation (15 mA for 10 ms at 4 Hz) blocked the 50 min uptake of 14C-NA by the heart to a greater degree than either factor separately. The release of noradrenaline was determined after perfusing hearts with 14C-NA followed by perfusion with solution containing pancuronium but no 14C-NA for 1 h. Pancuronium infusion did not significantly alter the release of 14C-NA from the heart after 1 h of perfusion. The infusion of pancuronium caused a reduction in both the rate and strength of myocardial contraction of the isolated heart which was reversed by perfusion with perfusate free of pancuronium. Following perfusion with pancurnium the rate and strength of contraction of the heart was seen to "rebound" above pre-pancuronium values for a short period. The rebound of myocardial rate and contraction may have been due to the presence of myocardial noradrenaline previously blocked from reuptake by pancuronium since hearts removed from reserpinized animals did not demonstrate "rebound."

Knockdown of canonical transient receptor potential proteins 1, 4, and 6 attenuates store‐operated calcium entry and calcium responses to acute hypoxia in pulmonary arterial smooth muscle cells

Acute hypoxia increases intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ entry through store-operated Ca2+ channels (SOCC) in myocytes from distal pulmonary arteries, which are thought to be the major locus of hypoxic pulmonary vasoconstriction. SOCC may be composed of canonical transient receptor potential (TRPC) proteins, and TRPC1, 4, and 6 are expressed in rat distal pulmonary arterial smooth muscle cells (PASMC). To determine if these TRPC contribute to store-operated Ca2+ entry (SOCE) and [Ca2+]i responses to hypoxia in these cells, we used fluorescence microscopy and the Ca2+-sensitive dye, Fura-2, to measure [Ca2+]i and SOCE in rat distal PASMC 24 h after treatment with siRNA targeted to TRPC1, 4, or 6, followed by exposureto normal basal medium for 24 h. As measured by real-time PCR and Western blotting, RNA interference decreased expression of targeted TRPC mRNA and protein by 75–95%, but did not decrease non-targeted TRPC expression. Compared to nontargeted siRNA, treatment with siRNA targeted to TRPC1, 4, or 6 caused 1) 54, 31, and 39% decreases in SOCE, as measured by quenching of Fura-2 fluorescence by Mn2+ at 10 min in cells perfused with Ca2+-free Kreb's Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid and nifedipine; and 2) 84, 44, and 41% decreases in the elevation of [Ca2+]i caused by perfusion with KRBS equilibrated with 4% O2-5% CO2 for 20 min; and 3) no decreases in elevation of [Ca2+]i in cells perfused with KRBS containing 60 mM KCl (equimolar substitution for NaCl) for 10 min. These results suggest that TRPC1, TRPC4, and TRPC6 all contribute to SOCE and [Ca2+]i responses to hypoxia in rat distal PASMC, but that TRPC1 is quantitatively more important than TRPC4 or TRPC6. (Supported by NIH grants: HL-75113 (J T Sylvester), HL-079981 (J Wang), HL-093020 (J Wang)).