, 1991). Hematology has been a valuable tool to diagnose many human diseases (Blaxhall and Daisley, 1973 and Heath, 1995), and is used in animals as well. In healthy fish, leukocytes are present in specific proportions and locations in body tissues. These cells orchestrate the initial line of defense against pathogens

(Stoskopf, 1993). The blood cells of fish PI3K inhibition are produced in hematopoietic tissues located in the spleen and kidney (Heath, 1995). Exposing fish to pollutants induces pathological changes in the kidney and liver (Adams et al., 2010 and Velmurugan et al., 2007). Leukocytopenia in fish is induced by many types of stress, and increases the susceptibility of fish to diseases (Razquin et al., 1990). Melano-macrophages are macrophages in which the cytoplasm contains RAD001 pigments such as lipofuscin, melanin, and hemosiderin, and melano-macrophage centers (MMCs) are aggregations of melano-macrophages in the stroma of hematopoietic tissues (Agius and Roberts, 2003). Melano-macrophage

centers are usually located close to a blood vessel in the spleen (Ferguson, 1976 and Graf and Schluens, 1979). The specific role of MMCs is not certain, but it is clear that they increase in size and number when fish are stressed or exposed to pathogens. Melano-macrophage centers are used as biomarkers for water quality and the health status of fish (Micale and Perdichizzi, 1990, Bucke et al., 1992 and Suresh, 2009), and can significantly increase in number and size during environmental contamination (Fournie et al., 2001), detoxification processes (Herraez and Zapata, 1991) and immunological responses (Wolke, 1992 and Agius and Roberts, 2003). Marine life is often exposed to pollutants from human activities,

and a few methods have been used routinely to determine environmental exposure (van der Oost et al., 2003). The common method measures the response of fish hepatic CYP450 1A activities (Whyte et al., 2000). Ethoxyresorufin-O-deethylase or EROD activity is performed by cytochrome P450 A1, an evolutionarily conserved enzyme involved in clearance of hydrocarbons. This enzyme is induced following exposure to hydrocarbons, such as those found in crude oil. It is an indicator that hydrocarbon exposure has occurred and is used as a monitoring tool for the health status of marine life and contamination Histone demethylase in water ( Straus et al., 2000). Each year, approximately 5 million metric tons of crude oil enter the aquatic environment from oil spills (Edwards et al., 2003). A direct link between oil exposure and increased bacterial or viral disease occurrence has not been determined. However, indirect evidence exists. Our study was conducted to determine the effects of oil exposure on the peripheral blood cells and tissues of Gulf of Mexico fish and utilized hematology, toxicology, and histology. Alligator gar (Atractosteus spatula), Gulf killifish (Fundulus grandis) and sea trout (Cynoscion nebulosus) were captured and sampled.

Therefore, and since at different food levels b did not differ significantly, a stronger curvature seems to be realistic for their copepod population. McLaren et al. (1969) suggested that thermal acclimation would only affect parameter α. If this is true, the different values of b may point to fundamental physiological differences between different populations of Temora. This is in contrast with the observation of those authors that b is constant within closely related species (see p. 82 in Klein Breteler & Gonzalez (1986)). The stage duration for each model stage (N1–N6 – naupliar stage, C1, C2, C3, C4, C5 – the five copepodid stages) and the generation

time using Bĕlehrádek’s function were obtained in the present work in accordance with the data of D (see

Figure 4 in Klein Breteler & Gonzalez (1986)). Here, the parameter b was taken from Klein Breteler & Gonzalez (1986); in addition, Selleckchem PF2341066 the values of α calculated in this paper vary from 2 to 3.5 and resemble the values of Klein Breteler & Gonzalez (1986). Bĕlehrádek’s function was converted to D = 10a(T − α)b, where the parameters a and b were described as a function of food concentration: α = a1 log Food + b1 and a = a2 log Food + b2 with the correlation coefficient from 0.69 to 0.97 for the naupliar stage (N1–N6) and the copepodid stage (C1–C5). But the correlation coefficient for a and α as a function of food concentration was too low for all copepodid stages separately (C1, C2, C3, C4, learn more C5). This meant that Bĕlehrádek’s function could not be used to define the mean development times for each copepodid stage separately. In view of this, the stage duration D in this work was obtained as a function of food concentration and temperature using the minimum development time Dmin. Dmin is the value for which the development rate is not Progesterone limited by food availability. The common logarithm of Dmin for T. longicornis was related linearly to the common logarithm of temperature: equation(1) logDmin=alogT+b. The values of a, b, and r, the correlation coefficients for developmental stages N1–N6, C1, C2, C3, C4 and C5 are given in Table 1. 96% of the values of Dmin

computed with equation (1) as a function of temperature lie within the range of the parameter Dmin given by Klein Breteler et al. (1982). The regression equations for each of the model stages of T. longicornis at temperatures ranging from 5 to 20°C are shown in Figure 1. The stage duration D of T. longicornis for developmental stages N1–N6, C1, C2, C3, C4 and C5, and for the period from N1 to medium adult was also obtained here. It was found to be very sensitive to changes in temperature and food concentration. Conversion of the data for D after Klein Breteler & Gonzalez 1986– see Figure 4 in this paper) to natural logarithms yielded a linear relationship between time and food concentration. This relationship was described by the equation equation(2) ln(D−Dmin)=aFood+b; hence, D=eaFood+b+Dmin.

Epochs including artifacts H 89 datasheet such as eye blinks and eye movements identified by visual

inspection were excluded from the analyses. To identify the sources of the evoked activities, ECD analyses were performed using commercial software (MEG 160; Yokogawa Electric Corporation). The ECDs with goodness of fit (GOF) values above 90% were used, based on a previous report (Bowyer et al., 2003, Dalal et al., 2008 and Sekihara and Nagarajan, 2008). Anatomical magnetic resonance imaging (MRI) was performed using a Philips Achieva 3.0TX (Royal Philips Electronics, Eindhoven, the Netherlands) to permit registration of magnetic source locations with their respective anatomical locations. Before MRI scanning, five adhesive markers (Medtronic Surgical Navigation Technologies Inc., Broomfield, CO) were attached to the skin of their head (the first and second ones were located at 10 mm in front of the left and right tragus, the third

at 35 mm above the nasion, and the fourth and fifth at 40 mm right and left of the third one). The MEG data were superimposed on MR images using information obtained from these markers and MEG localization coils. The PFS is Tanespimycin research buy a questionnaire comprised of 15 items scored on a 5-point Likert-type scale ranging from 1 (Do not agree at all) to 5 (Strongly agree), with higher scores indicating greater responsiveness to food environment ( Lowe et al., 2009). Based on previous factor analyses, the PFS has been shown

to contain a three-factor structure of food proximity consisting of: (1) ‘food available’, which describes the reaction when food is not physically present but is always available; (2) ‘food present’, which characterizes the reactions to palatable foods when they are physically present, but have not yet been tasted; and (3) ‘food tasted’, which characterizes the reactions to palatable foods when first tasted, but not yet consumed. According to previous studies using the PFS ( Yoshikawa et al., 2012, Cappelleri et al., 2009 and Schultes et al., 2010), the subscale scores for PFS are calculated as the average scores of all items included in each subscale (ranged 1–5) as well DNA ligase as aggregated factor scores as the average scores of all 15 items (ranged 1–5). The participants completed the PFS before the MEG recordings. Data are expressed as mean±SD unless otherwise stated. Subjective levels of appetitive motives during the MEG recordings were compared between the Fasting and ‘Hara-Hachibu’ condition by paired-t test. All the MEG variables under four conditions (food images in the Fasting condition, mosaic images in the Fasting condition, food images in the ‘Hara-Hachibu’ condition, and mosaic images in the ‘Hara-Hachibu’ condition) were compared using two-way ANOVA for repeated measures. A paired t-test was used to evaluate significant differences between the two conditions.

Investigators obtained informed, written consent from each patient and/or the parent or guardian. Patients were randomized to receive taliglucerase alfa 30 U/kg selleckchem or 60 U/kg per infusion every other week for 12 months.

The primary end point was the median percent change in hemoglobin concentration from baseline and the interquartile range of median percent change in hemoglobin levels from baseline. Secondary end points included the percent changes from baseline in spleen volume, liver volume, platelet counts, and either chitotriosidase or CCL18 activity. Exploratory end points of organ volumes expressed as multiples of normal (MN) were calculated using normal spleen volume = 2 mL/kg multiplied by body weight in kg and normal liver volume = 25 mL/kg multiplied by body weight in kg. Exploratory end points included: change in height, weight, puberty, and bone age (based on radiograph of the left hand and wrist); occurrence of bone events including bone crises; and quality of life using Child Health Questionnaire™ (CHQ) PF-28 (valid for patients aged 5 to 18 years). Safety end points included AEs and changes in clinical laboratory

findings, echocardiographic readings, and anti-taliglucerase alfa antibody titers. Occurrence of bone events, including bone crises, was part of the analysis of AEs. Male and female patients aged 2 to < 18 years were required to have a diagnosis of GD with leukocyte acid beta-glucocerebrosidase activity level ≤ 30% of the mean activity of the reference range for healthy individuals. Patients were eligible if they had not Vemurafenib in vivo received ERT in the past or within the previous 12 months and had a negative anti-glucocerebrosidase assay assessment, had not received substrate reduction therapy for GD in the past 12 months, and were judged in need of treatment with ERT based on clinical condition and the opinion of the local investigator. Gefitinib Patients were excluded based on any of the following criteria: presence of complex neuronopathic features other than longstanding

oculomotor gaze palsy; unresolved anemia due to iron, folic acid, or vitamin B12 deficiency; currently taking another investigational drug for any condition; previous hypersensitive reaction to alglucerase or imiglucerase; history of allergy to carrots; inability of parents or guardians to understand the nature, scope, and consequences of study participation; and presence of any medical, behavioral, psychological/emotional condition that would, in the investigator’s opinion, interfere with full participation in the study. Spleen and liver volumes were measured using magnetic resonance imaging as previously reported [15] and were assessed at BioClinica, Lyon, France. Beta-glucocerebrosidase activity, chitotriosidase or CCL18 activity, and DNA sequencing were performed at a centralized laboratory, the Academic Medical Center in Amsterdam, The Netherlands.

These magnets are for scientific research on fundamental medical and physiological problems ranging from cognitive science to aging, heart disease and cancer. The opportunities opened by much higher magnetic fields than exist today are tremendous as many human health conditions cannot be approached by any other methods as discussed in the body of this chapter. The technologies focused here upon are initially meant for research and not for routine clinical use. At a clinical level, ca. 40,000 clinical systems will have been installed worldwide

click here by 2013. The majority of new installations are for 1.5 T (64%) with the remainder equally divided between 3.0 T and less than 1.5 T. The current distribution of field strengths for magnets at or above 7 T is approximately as follows: 50 at 7 T, 5 at 9.4 T, 2 at 10.5 T, 1 at 11.7 T. One 14 T for human brain imaging is being funded for see more South Korea. Animal research systems with small bores and high field are also in increasing demand world wide. As discussed in previous sections, the field of magnetic resonance spectroscopy (NMR, MRS) is now of major importance particularly to chemistry. In

1972, chemist Paul Lauterbur showed that one can image the spatial distribution of the hydrogen nucleus concentration (mainly water) in objects and this led to magnetic resonance imaging [16]. Magnetic resonance imaging (MRI) initially, and years later functional magnetic resonance imaging (fMRI), eventually became major modalities for research and diagnostic medicine as well as for animal physiology studies – particularly since the mid 1980s. NMR spectroscopy and MRI have followed parallel paths of technological development,

despite their differences in fields and sample sizes (Fig. 1). Throughout the development of MRI and MRS, each substantive increase in field strength has in time led to dramatic improvements in the quality of images and spectra obtainable, and usually to ‘quantum leaps’ in the information available about tissue structure and function (Fig. 2). Each major increase Methocarbamol in field has also introduced new technical challenges and problems that have required creative scientific and engineering solutions in order to realize the potential to improve image quality [18]. The evolution of higher field systems has continued. By 1988 success in development of a whole body 4 T system was reported [19], [20], [21] and [22] and commercial vendors made a small number of 4 T MRI magnets. However, ultimately the main industrial effort focused on developing scanners operating at 3 T, and these systems are replacing 1.5 T systems in many clinical applications. Much of the early 3 T developments emphasized brain imaging, partly motivated by the discovery of the benefits of blood oxygenation level-dependent susceptibility contrast as a measure of brain activity. This phenomenon is also known as functional MRI (fMRI).

Furthermore, by choosing our study period, we have ensured no systematic changes in coding because the ICD-10 coding system has been in continuous use in HES from 1995 to present. This, of course, does not exclude variation in rates of coding over the study period affecting our

estimates. For example, if the potential error in coding was systematically changing over time with increased coding of patients’ comorbidity rather than patients having more comorbidity, then clearly that could bias our results. However, the different trends VX-809 in comorbidity for variceal and nonvariceal bleed admissions and different trends in mortality in different age and comorbidity strata suggest that there was no systematic change in comorbidity coding over the time period of our study. Under-reporting of the comorbidities in the Charlson index may have resulted in incomplete adjustment for comorbidity. However, although the alternative Elixhauser index assessed almost twice the number of comorbidities, it did not alter the adjustment of comorbidity in the

model. Comorbidity adjustment by either index increased the magnitude of the mortality reduction, and, therefore, any residual confounding in this regard would only, we believe, cause an underestimate of the real mortality trend in our study. A PubMed search, to October 2010, found the largest comparable population-based study for nonvariceal hemorrhage mortality trends used a Canadian hospital discharge database with ICD-10 and ICD-9 codes. However, it identified less than one-third of the number of bleeds used for Selleckchem DAPT this study (n = 142,363) and was not able to identify a reduction in case fatality for nonvariceal hemorrhage between 1993 Mirabegron and 2003.3 The researchers adjusted for changes in age but not for changes in comorbidity. They

also only identified deaths that occurred before discharge. The low mortality identified in this study (3.5%) is similar to other North American20 and Mediterranean1 and 21 studies but is much lower than other European studies.2, 22 and 23 However, a study of Medicare patients in the United States found that the proportion being managed as outpatients varied between states from 18.6% to 45.3%.24 These differences in practice would lead to differences in inpatient study populations and confound comparisons with countries such as England where outpatient management is not routine. Although the most recent report from the US National Inpatient Sample showed a 23% reduction in upper gastrointestinal hemorrhage mortality from 1998 to 2006 (n = unreported because only extrapolated estimates from the 20% sample are provided),20 this was a global figure for the reduction seen at the end of the study rather than year on year, and it did not distinguish variceal and nonvariceal hemorrhage. Another report from the US National Inpatient Sample noted an adjusted reduction in variceal hemorrhage from 18% to 12%.

Segregation of Dabrafenib price the IPL areas was driven mainly by differences in the densities

of GABAA, α2 and α1 receptors. In the right hemisphere (Fig. S2), only the areas of the Broca region (44d, 44v, 45a, 45p and IFS1/IFJ) cluster together and are separated from the mouth motor representation area 4v, the prefrontal area 47 and the temporal areas pSTG/STS and Te2. This segregation was due mainly to differences in M2, 5-HT2 and NMDA receptor densities, and may reflect a difference between the language dominant left hemisphere and the right hemisphere. Areas 7, 9, 46, 32, FG1 and FG2 build a separate cluster in the left hemisphere (Fig. 4) and have been demonstrated to be involved in a variety of cognitive functions. Although area 46 was described as being part of a language processing network (Turken & Dronkers, 2011), while area

9 was demonstrated to be involved in idiom comprehension (Romero, Walsh, & Papagno, 2006) and in fronto-temporal interactions for strategic inference processes during language comprehension (Chow, Kaup, Raabe, & Greenlee, 2008), both are also involved, as is area 7, in the neural network associated with working memory, planning, and reasoning-based click here decision making (D’Esposito et al., 2000, Levy and Goldman-Rakic, 2000 and Marshuetz et al., 2000). Interestingly, deactivations of left areas 9 and 46 were found to

correlate with activations of left area 32 during a task involving the processing of self-reflections during decision making (Deppe, Schwindt, Kugel, Plassmann, & Kenning, 2005). Although areas 46 and 9 are involved in language and memory processes, the fact that their receptor fingerprints build a cluster with those of other areas involved in memory functions (areas 7 and 32; Garn et al., 2009, Hernandez et al., 2000, Kan and Thompson-Schill, 2004 and Whitney et al., 2009) may highlight the preferential involvement of the prefrontal areas 46 and 9 in memory-related processes. The extrastriate visual areas FG1 and FG2 are associated mafosfamide with cognitive functions such as word form (left hemisphere) and face (right hemisphere) recognition, visual attention, and visual language perception (Caspers et al., 2013b and Dehaene and Cohen, 2011). Although some of the IPL areas of the left hemisphere may belong to the functionally defined wider Wernicke region, they differ from 44v, 44d, 45a, 45p, IFS1/IFJ, and pSTG/STS in that they are not necessarily activated during sentence comprehension, but during semantic expectancy, preferentially in degraded speech (Obleser and Kotz, 2010 and Obleser et al., 2007) and in semantic and phonological processing (Gernsbacher and Kaschak, 2003, Geschwind, 1970 and Price, 2000).

É convicção dos autores que os dados obtidos não diferem significativamente do que se verificará na globalidade dos hospitais portugueses. A população estudada pertence a uma faixa etária avançada, o que corrobora o observado atualmente na generalidade das enfermarias hospitalares. De acordo

com diversos trabalhos publicados na literatura, a idade e a existência de comorbilidades buy INCB024360 constituem justamente importantes fatores de prognóstico adverso neste grupo de doentes.11, 12 and 13 O foco séptico abdominal foi o mais frequente, o que está de acordo com o expectável num serviço de gastrenterologia. Ainda assim, existe um número considerável de infeções com outra localização, correspondendo na sua maioria a doentes com cirrose hepática descompensada. O raro internamento na UCIGH contrasta com a elevada proporção de casos com hipoperfusão ou choque séptico, próxima dos 50%. É precisamente este subgrupo de doentes que apresenta risco de mortalidade acrescido, beneficiando de uma abordagem mais precoce e agressiva, de acordo com as recomendações da SSC8. O número limitado de vagas desta unidade terá certamente contribuído para esta disparidade,

ainda assim este constitui um aspeto passível de alguma otimização Sorafenib cost futura. De acordo com os resultados deste estudo, a monitorização e avaliação de sinais de falência de órgão é deficitária. Analisando de forma mais pormenorizada os valores encontrados, Oxymatrine é de salientar a ausência de avaliação/registo da gasometria arterial com lactatos, algaliação e débito urinário num elevado número de casos. No contexto de sépsis, todos estes são parâmetros fundamentais de monitorização, podendo traduzir falência orgânica e a necessidade de intervenções terapêuticas específicas. Tivessem sido corretamente reconhecidos os casos de hipoperfusão e aplicadas as recomendações vigentes, estaria indicada a obtenção de um acesso venoso central num maior número de doentes, para avaliação da pressão e

saturação venosas centrais e adequado manejo da administração de fluidos e vasopressores, o que não se verificou. Estes dados devem ser interpretados com cautela. Obviamente, apenas foi possível avaliar os parâmetros registados, pelo que os valores obtidos poderão ser fruto de registos incompletos e não necessariamente do défice de avaliação. Além disso, nos doentes com tempo de permanência no SU mais curto a abordagem poderá ter sido repetida e complementada na enfermaria de destino. A identificação do foco de infeção e dos microrganismos envolvidos é um passo primordial na abordagem do doente séptico, embora sempre sem prejuízo da instituição das medidas terapêuticas prioritárias. Os exames a efetuar em cada situação estão, naturalmente, dependentes do quadro clínico e do contexto de cada doente.

To determine the viability of cells exposed to MWNT-7, we performed an AB assay (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cells were incubated for 24 h at 37 °C in 0.1 ml of culture medium with various concentrations of MWNT-7 in 96-well culture plates.

The control cells were cultured in the culture medium containing dispersant. Viable cells metabolized the dye, which resulted in an increase in the fluorescence intensity, as determined by excitation/emission at 550/600 nm on a fluorescence multiplate reader (PowerScan 4, DS Pharma Biomedical, Osaka, selleck chemicals Japan). Cytotoxic activity was calculated as follows: percent cytotoxicity = 100 × experimental value/control value. Test media were assayed 6 times. To determine the effect of endocytosis inhibitors, cells cultured on 96-well culture plates for 24 h were pretreated with chlorpromazine hydrochloride (20 μM; Nacalai) dissolved in PBS or indomethacin (50 μM; SIGMA, St. Louis, MO, USA) dissolved in ethanol for 15 min. The

cells were then exposed to MWNT-7 (50 μg/ml) with the inhibitors for 2 h. The cells were washed Navitoclax clinical trial twice with Dulbecco’s PBS (DPBS) at 4 °C and cultured in each medium without MWNT-7 or the inhibitors for 22 h. Thereafter, the cells treated with the AB reagent were assayed. Cells were cultured on ibiTreat dishes (μ-dish35 mm high; ibidi GmbH, Martinsried, Germany) for 24 h in a 5% CO2 incubator. The cells were then incubated with or without MWNT-7 (1 μg/ml) for 24 h. Prior to observation, the cells were washed twice and stained with bisbenzimide H33342 fluorochrome

trihydrochloride (H33342, Liothyronine Sodium 1 μg/ml; Nacalai) for 30 min. The cells were visualized using differential interference contrast (DIC) and fluorescence by fluorescence microscopy (AxioObserverZ1, Zeiss, Jena, Germany) in a 5% CO2 chamber at 37 °C using a 40× objective. To determine the effect of endocytosis inhibitors, cells cultured on ibiTreat dishes for 24 h were pretreated with 2 types of endocytosis inhibitors for 15 min and then exposed to MWNT-7 (10 μg/ml) and H33342 for 2 h. The cells were washed twice with DPBS at 4 °C and observed in each medium without MWNT-7 or the inhibitors. We previously have reported that certain cytokines as secreted as part of the inflammatory response in BEAS-2B cells exposed to MWCNTs (Tsukahara and Haniu, 2011). Although the secretion of interleukin (IL)-6 and IL-8 was shown to increase upon exposure to MWCNTs, other cytokines (IL-12, TNF-α, IL-10, and IL-1β) were not detected. Therefore, we selected IL-6 and IL-8 for evaluation in this study. Cytokines in the culture supernatant were measured using a cytometric bead array flex set system (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s protocol.

Several abnormal, or abnormally regulated, cation transporters participate in the pathogenesis of SCD [13], [14] and [15]. These include the K+–Cl− cotransporter (or KCC) and the Ca2 +-activated K+ channel (or Gardos channel), transport systems whose molecular identities are established. A third pathway, sometimes termed Psickle, is activated by HbS polymerisation

and RBC shape change [13] and [16]. Psickle is thought to function predominantly as a deoxygenation-induced cation pathway. Although it remains enigmatic at a molecular level, Psickle will allow entry of Ca2 +[17] and [18], and loss of Mg2 +[19] and [20], with subsequent activation of the Gardos channel and perhaps KCC. The three pathways interact to mediate solute loss [15], thereby concentrating HbS, which greatly reduces Tyrosine Kinase Inhibitor Library the CT99021 mouse lag time for polymerisation upon deoxygenation—hence increasing the likelihood of sickling and ischaemia in the microvasculature. In this report, radioactive tracer methodologies have been used to investigate the effects of ortho (o)-vanillin on K+ permeability, KCC, the Gardos channel and Psickle in RBCs from SCD patients. Results show that this aromatic aldehyde markedly inhibits all three, as well as also affects HbS polymerisation and sickling, but also stimulates an unidentified K+ efflux pathway. These additional

actions of o-vanillin may be of significant consideration when designing similar compounds to ameliorate the complications of SCD. Bumetanide, 3-[N-morpholino] propane sulfonic acid (MOPS), N-ethylmaleimide, ouabain, ortho (o)-vanillin, and salts were purchased from Sigma Chemical Co. (Poole, Dorset, UK). Clotrimazole and A23187 were purchased from Calbiochem (Nottingham, UK). 86Rb+ was supplied by Perkin Elmer (Beaconsfield, UK). Blood samples were obtained by venepuncture

of patients with sickle cell disease (SCD), both homozygous HbSS and heterozygous HbSC, with permission under ethical consent, using the anticoagulant EDTA. Samples were kept at 4 °C until use within 48 h. The standard saline (MBS) comprised (in mM): 145 NaCl, 1.1 CaCl2, 5 glucose and 10 MOPS, (pH 7.4 at 37 °C; 290 ± 5 mOsmol kg −1 H2O). Megestrol Acetate For experiments in which Cl− dependence of K+ influx was examined, NO3−-containing salts replaced those containing Cl−. To prevent the rapid RBC shrinkage which would otherwise occur following maximal stimulation of the Gardos channel in experiments in which intracellular Ca2 + was directly raised by incubation with the Ca2 + ionophore A23187, a high-K+- and low-Ca2 +-containing saline was used, comprising (in mM): 80 KCl, 70 NaCl, 0.01 CaCl2, 0.15 MgCl2, 5 glucose and 10 MOPS. The wash solution to remove unincorporated 86Rb+ comprised isotonic MgCl2 (107 mM), buffered with MOPS (10 mM), pH 7.4 at 4 °C. Stock solutions of bumetanide (10 mM) were prepared in 100 mM Tris base and used at a final concentration of 10 μM. Stock solutions of ouabain (10 mM) were prepared in distilled water and used at a final concentration of 100 μM.