Ringer Medium Research Articles

Electropolymerization of functionalized barium titanate reinforced polypyrrole composite coatings on nitinol alloy for biomedical applications

In the current study, the nitinol alloy (NiTi) was electrochemically coated with conductive polypyrrole (PPy) and polypyrrole/polydopamine functionalized barium titanate nanoparticles (PBT). The composite coatings were attained by adding different concentrations of PBT nanoparticles (1–10 wt%) to the electrochemical deposition bath. The results of transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), dynamic laser scanning (DLS), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) confirmed the successful development of a polydopamine (PDA) layer on the surface of barium titanate nanoparticles (BT NPs) and their incorporation into PPy matrix composite coatings. The optimum concentration of PBT was beneficial for coating adhesion strength due to PDA bonding with PPy and metallic substrate via the catechol moieties. Electrochemical examinations demonstrated that PPy/5PBT coating revealed excellent corrosion protection in the Ringer medium. The PBT particles also reduced the overall conductivity of the PPy coating by about tenfold and significantly improved the mineralization of the composite coatings by providing favorable sites for Ca ion adsorption and subsequent hydroxyapatite (HA) formation reactions. The results of osteoblast cell attachment revealed that PBT had a promising effect on the cellular behavior of the composite-coated samples.

Fresh and cultured mouse islets differ in their response to nutrient stimulation.

Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be typical for mouse islets. This was also the response of cultured islets to the nutrient secretagogue alpha-ketoisocaproic acid, whereas the secretion of fresh islets increased similarly fast but remained strongly elevated. The responses of fresh and cultured islets to purely depolarizing stimuli (tolbutamide or KCl), however, were closely similar. Signs of apoptosis and necrosis were rare in both preparations. In cultured islets, the glucose-induced rise of the cytosolic Ca2+ concentration started from a lower value and was larger as was the increase of the ATP/ADP ratio. The prestimulatory level of mitochondrial reducing equivalents, expressed as the NAD(P)H/FAD fluorescence ratio, was lower in cultured islets, but increased more strongly than in fresh islets. When culture conditions were modified by replacing RPMI with Krebs–Ringer medium and FCS with BSA, the amount of released insulin varied widely, but the kinetics always showed a predominant first phase. In conclusion, the secretion kinetics of fresh mouse islets is more responsive to variations of nutrient stimulation than cultured islets. The more uniform kinetics of the latter may be caused by a different use of endogenous metabolites.

AP-2-dependent Internalization of Potassium Channel Kir2.3 Is Driven by a Novel Di-hydrophobic Signal

The localization and density of Kir2.3 channels are influenced by the balance between PDZ protein interaction at the cell surface and routing into the endocytic pathway. Here, we explore mechanisms by which the Kir2.3 channel is directed into the endocytic pathway. We found that Kir2.3 channels are constitutively internalized from the cell surface in a dynamin-dependent manner, indicative of vesicle-mediated endocytosis. The rate of Kir2.3 endocytosis was dramatically attenuated following RNA interference-mediated knockdown of either alpha adaptin (AP-2 clathrin adaptor) or clathrin heavy chain, revealing that Kir2.3 is internalized by an AP-2 clathrin-dependent mechanism. Structure-rationalized mutagenesis studies of a number of different potential AP-2 interaction motifs indicate that internalization of Kir2.3 is largely dependent on a non-canonical di-isoleucine motif (II413) embedded within the C terminus. Internalization assays using CD4-Kir2.3 chimeras demonstrate that the di-isoleucine signal acts in an autonomous and transplantable manner. Kir2.3 co-immunoprecipitates with alpha adaptin, and disruption of the di-isoleucine motif decreased interaction of the channel with AP-2. Replacement of the di-isoleucine motif with a canonical di-leucine internalization signal actually blocked Kir2.3 endocytosis. Moreover, in yeast three-hybrid studies, the Kir2.3 di-isoleucine motif does not bind the AP-2 alphaC-sigma2 hemicomplex in the way that has been recently observed for canonical di-leucine signals. Altogether, the results indicate that Kir2.3 channels are marked for clathrin-dependent internalization from the plasma membrane by a novel AP-2-dependent signal.

4α‐PDD induces Ca2+ influx in human corneal epithelial cells by activating TRPV4 channels

Abstract Purpose: Transient receptor potential (TRP) isoform expression is evident in human corneal epithelial cells (HCEC‐SV40). However, their role in maintaining corneal epithelial homeostasis is not fully understood. We probed for gene and protein expression as well as functional activity of the vanilloid subtype, TRPV4, in immortalized HCEC‐SV40 since they elicit Ca2+ dependent regulatory volume decrease (RVD) responses during exposure to a hypotonic challenge. Methods: RT‐PCR and Western blotting analyses identified TRPV4 gene and protein expression. Functional activity was assessed based on determining whether the TRPV4 selective agonist, 4α‐PDD, induced transients increases in intracellular Ca2+ concentration. Results: Single cell fluorescence imaging results showed that 4α‐PDD (3 μM) increased intracellular Ca2+ concentration. The fura2 fluorescence ratio (f340/f380) was 0.39 ± 0.03578 in the resting state (n = 5). After application of 4α‐PDD it increased to 0.904 ± 0.14363 (n = 5; p = 5.72077×10‐5). This increase was abolished by the TRP channel blocker ruthenium red or by Ca2+‐free Ringer's medium. Conclusions: In conclusion, there is functional TRPV4 expression in HCEC‐SV40. TRPV4 expression may provide an osmosensor role to induce RVD behavior during exposure to a hypotonic challenge since this response is mediated through intracellular Ca2+ transients.Supported in part DFG Pl 150/14‐1 and NIH, EY04795. CR: none

Cadherin Conformations Associated with Dimerization and Adhesion

To investigate conformations of C-cadherin associated with functional activity and physiological regulation, we generated monoclonal antibodies (mAbs) that bind differentially to monomeric or dimeric forms. These mAbs recognize conformational epitopes at multiple sites along the C-cadherin ectodomain aside from the well known Trp-2-mediated dimer interface in the N-terminal EC1 domain. Group 1 mAbs, which bind monomer better than dimer and the Trp-2-mutated protein (W2A) better than wild type, recognize epitopes in EC4 or EC5. Dimerization of the W2A mutant protein via a C-terminal immunoglobulin Fc domain restored the dimeric mAb-binding properties to EC4-5 and partial homophilic binding activity but did not restore full cell adhesion activity. Group 2 and Group 3 mAbs, which bind dimer better than monomer and wild type better than W2A, recognize epitopes in EC1 and the interface between EC1 and EC2, respectively. None of the mAbs could distinguish between different physiological states of C-cadherin at the cell surface of either Xenopus embryonic cells or Colo 205 cultured cells, demonstrating that changes in dimerization do not underlie regulation of adhesion activity. On the cell surface the EC3-EC5 domains are much less accessible to mAb binding than EC1-EC2, suggesting that they are masked by the state of cadherin organization or by other molecules. Thus, the EC2-EC5 domains either reflect, or are involved in, cadherin dimerization and organization at the cell surface.

A comparative study of bioradiography in human brain slices and preoperative PET imaging

Novel autoradiography (bioradiography) images in human neocortical brain slices which were obtained at operation from seven patients with intractable epilepsy who had received a 2-[ 18F]fluoro-2-deoxy- d-glucose-positron emission tomography (FDG-PET) examination preoperatively, were acquired in Krebs–Ringer medium (control condition) and that with high K + (high K + condition) containing FDG and compared with FDG-PET uptake. The FDG uptake images in rat brain slices were also acquired as a reference and compared with that in humans. In the slices incubated under high K +, FDG uptake in both human and rat gray matter was significantly enhanced, whereas that in the white matter was not. But the variance of uptake was larger in humans than the rats. This might indicate the different degree of progress of epilepsy in the sampled brain tissues. The uptake rates of FDG in human gray matter under the control condition showed an inverse correlation with those seen in PET, which were evaluated as sampled and contralateral gyri (SG/CG) and sampled gyri and cerebellar cortex (SG/CB) ratio. On the contrary, it showed a weak positive correlation with PET under the high K + condition. The uptake rates of FDG in human gray matter expressed as a high K +/control ratio, closely matched that observed by FDG-PET, which were evaluated as the SG/CG ratio and the SG/CB ratio. Our experimental system provides useful information for the interpretation of PET data in epileptics and the theoretical basis to interpret the results of metabolic studies using living human brain tissues for further use in pharmacological manipulation.

In vivo neurochemical characterization of Anatoxin-a evoked dopamine release from striatum

Anatoxin-a (AnTx) is a natural neurotoxin, which acts as a potent and stereoselective agonist at the nicotinic acetylcholine receptors. The in vivo actions of the AnTx on dopamine (DA) release are scarcely characterized. The aim of this study was to determine the neurochemical bases for AnTx-induced striatal DA release, using the brain microdialysis technique, in freely moving rats. Local application of AnTx (3.5 mM) through the microdialysis probe produced an increase in striatal DA levels (701 +/- 51% with respect to basal values). The effect of infusion of AnTx in Ca(2+)-free Ringer medium, in Na(+)-free Ringer medium and with TTX in the medium, was inhibited. Also, reserpine pre-treatment blocked the action of AnTx on striatal DA levels. To investigate the involvement of the DA transporter, the effects of AnTx were observed in the presence of nomifensine. The coadministration of AnTx and nomifensine evoked an additive effect on striatal DA levels. The latter results show that the DA release is not mediated by a decreased DA uptake. Taken as a whole, these results suggest that the effects of AnTx are predominantly mediated by an exocytotic mechanism, Ca(2+)-, Na(+)- and TTX-dependent, and not by a mechanism mediated by the DA transporter.

Tribocorrosion of Aisi 304l Stainless Steel under Continuous Sliding in Ringer's Medium

not Available.

Study of the effect of aqueous extract of Grewia tenax fruit on iron absorption by everted gut sac

Grewia tenax roots, leaves, juice and fruit decoctions have been used in Africa and Southeast Asiatic countries for a variety of medical purposes. In this investigation, we report the effect of aqueous extract of Grewia tenax fruit (AEGTF) on the variation in vitro of iron absorption. The incubation of freshly prepared rat everted gut sac (EGS) in Ringer medium containing FeSO 4 in the absence of AEGTF showed that in stomach there is iron absorption only at 15 min of incubation time, whereas, at duodenum and jejunum, iron uptake occurs just after 1 min of incubation time and the maximum of iron absorption is registered at 15 min of incubation time. Addition of AEGTF at different concentrations favors significantly this iron transfer from the mucous side toward the serous one. The maximum of iron absorption was carried out in the presence of AEGTF at 10 mg/ml and 5 min of incubation time in stomach, duodenum and jejunum. AEGTF used at high doses (20 and 30 mg/ml) reduced significantly iron uptake suggesting a probable toxic effect of this extract. Histological studies confirmed the presence of cytotoxic signs as multinucleated giant cells and the disappearance of enterocyte border brush. With the aim of elucidating the mechanism of action of AEGTF, we are attempting to isolate the active principles present in this extract.

Glucose triggers protein kinase A-dependent insulin secretion in mouse pancreatic islets through activation of the K+ATP channel-dependent pathway

To assess the significance of protein kinase A (PKA) in glucose triggering of ATP-sensitive K(+) (K(+)(ATP)) channel-dependent insulin secretion and in glucose amplification of K(+)(ATP) channel-independent insulin secretion. Insulin release from cultured perifused mouse pancreatic islets was determined by radioimmunoassay. In islets cultured at 5.5 mmol/l glucose, and then perifused in physiological Krebs-Ringer medium, the PKA inhibitors, H89 (10 micromol/l) and PKI 6-22 amide (30 micromol/l) did not inhibit glucose (16.7 mmol/l)-induced insulin secretion, but inhibited stimulation by the adenylyl cyclase activator, forskolin (10 micromol/l). In the presence of 60 mmol/l K(+) and 250 micromol/l diazoxide, which stimulates maximum Ca(2+) influx independently of K(+)(ATP) channels, H89 (10 micromol/l) inhibited Ca(2+)-evoked insulin secretion, but failed to prevent glucose amplification of K(+)(ATP) channel-independent insulin secretion. In the presence of 1 mmol/l ouabain and 250 micromol/l diazoxide, which cause modest Ca(2+) influx, glucose amplification of K(+)(ATP) channel-independent insulin secretion was observed without concomitant Ca(2+) stimulation of PKA activity. In islets cultured at 16.7 mmol/l glucose, glucose (16.7 mmol/l)-induced insulin secretion in physiological Krebs-Ringer medium was augmented and now inhibited by H89 (10 micromol/l), implicating that culture at 16.7 mmol/l glucose may increase Ca(2+)-sensitive adenylyl cyclase activity and hence PKA activity. In accordance, Ca(2+)-evoked insulin secretion at 60 mmol/l K(+) and 250 micromol/l diazoxide was improved, whereas glucose amplification of K(+)(ATP) channel-independent insulin secretion was unaffected. Glucose may activate PKA through triggering of the K(+)(ATP) channel-dependent pathway. Glucose amplification of K(+)(ATP) channel-independent insulin secretion, on the other hand, occurs by PKA-independent mechanisms.

Ethical and psychosocial issues related to interferon alpha therapy for chronic hepatitis C

The initial rate of net Mg2+ efflux was measured in human red blood cells by atomic absorption. In fresh erythrocytes incubated in Na+,K+-Ringer's medium this rate was 7.3 ± 2.8 μmol/I cells per h (mean±S.D. of 14 subjects) with an energy of activation of 13 200 cal/mol. Cells with total Mg2+ contents ([Mg]i) ranging from 1.8 to 24 mmol/l cells were prepared by using a modified p-chloromercuribenzenesulphonate method. Mg2+ efflux was strongly stimulated by increases in [Mg]i and in external Na+concentrations ([Na]o). A kinetic analysis of Mg2+ efflux as a function of [Mg]i and [Na]o revealed the existence of two components: (i) an Na+-stimulated Mg2+ efflux, which exhibited a Michaelian-like dependence on free internal Mg2+ content (apparent dissociation constant = 2.6 ± 1.4 mmol/l cells; mean ± S.D. of six subjects) and on external Na+ concentration (apparent dissociation constant = 20.5 ± 1.9 mM; mean ± S.D. of four subjects) and a variable maximal rate ranging from 35 to 370 μmol/l cells per h, and (ii) an Na+-independent Mg2+ efflux, which showed a linear dependence on internai Mg2+ content with a rate constant of (6.6 ± 0.7) · 10−3 h−. Fluxes catalyzed by the Na+-stimulated Mg2+ carrier were partially dependent on the ATP content of the cells and completely inhibited by quinidine (IC50 = 50 μM) and by Mn2+ (IC50 = 0.5–1.0 mM).

Assessment of choline uptake for the synthesis and release of acetylcholine in brain slices by a dynamic autoradiographic technique using [ 11C]choline

The uptake of choline for the synthesis and release of acetylcholine was investigated in brain slices by dynamic positron autoradiography using [ 11C]choline. Brain slices (330 μm) were incubated with [ 11C]choline in oxygenated Krebs–Ringer medium at 34 °C and serial two-dimensional time-resolved images of the uptake and release of radioactivity were recorded on Storage Phosphor screens. [ 11C]Choline uptake increased with the period of incubation and was 1.9 times higher in the striatum than cerebral cortex. The uptake in the striatum was significantly diminished by hemicholinium-3 (HC-3), an inhibitor of high-affinity choline uptake. Pretreatment of brain slices with 50 mM K + for 20 min enhanced the uptake in striatum. The uptake of [ 11C]choline in brain slices was saturable using nonlabeled choline. Two uptake systems, a high-affinity and a low-affinity system, were confirmed to exist by kinetic analysis using Lineweaver–Burk plots. The 11C radioactivity that had accumulated in the striatum disappeared on treatment with veratridine, a depolarization agent, in the presence of HC-3. This pattern of disappearance was consistent with that of the appearance of unlabeled and labeled acetylcholine in the medium. These results indicate that this method is useful for obtaining information regarding the uptake of choline for the synthesis and release of acetylcholine in live brain tissues.

Sodium-dependent and -independent choline uptake by type II epithelial cells from rat lung.

The uptake of 3H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 microM and 5.0 microM medium choline; at 5 microM, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 microM), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na+ was replaced by Li+, K+ or Mg2+, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na+ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na+ concentration. Kinetic properties of the uptake of 0.1 microM 3H-choline in the presence and absence of medium Na+ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5-100 microM) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na+; a Na(+)-dependent portion (a mean of 0.52 of the total) had a K(m) for choline of 1.5 microM while K(m) in the absence of Na+ (Li+ substituting) was 18.6 microM. (b) Inhibition by HC-3 (0.3-300 microM) gave Ki values of 1.7 microM and 5.0 microM HC-3 for the Na(+)-dependent and -independent fractions. The apparent K(m) of the Na(+)-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K(m) values reported for high-affinity, Na(+)-dependent choline uptake by neuronal cells.

Lithium regulation of protein phosphorylation in rat cerebral cortex slices in vitro.

The aim of the present study was to evaluate the direct effect of Li+ and Na+-Li+ exchange on protein phosphorylation in rat cerebral cortex slices incubated in Krebs Ringer medium. When Na+ concentration was varied in the incubation medium, either by replacement with Li+ or sucrose, a variable effect on [32P] phosphate incorporation into proteins was observed. Protein phosphorylation in cerebral cortex slices was very low in the absence of Na+, and some dependence of phosphorylating system of neural tissue on extra cellular concentration of Na+ was evident. Lithium was not able to replace sodium as far as protein phosphorylation in cortical slices is concerned. Ouabain was more effective in a Li+ containing medium in inhibiting protein phosphorylation, presumably due to improper functioning of the sodium pump.

The effects of near-UV radiation on elasmobranch lens cytoskeletal actin

The role of near-UV radiation as a cytoskeletal actin-damaging agent was investigated. Two procedures were used to analyse fresh smooth dogfish ( Mustelus canis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringer's medium, and with or without exposure to a near-UV lamp (emission principally at 365 nm; irradiance of 2·5 mW cm −2). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purified polymerized rabbit muscle actin were exposed to the same UV conditions and depolymerization was assayed by ultracentrifugation and high-pressure liquid chromatography. While the two actins studied do differ very slightly in some amino acid sequences, they would react physically nearly identically. The results showed that dogfish lenses developed superficial opacities due to near-UV exposure. Whole mounts of lens epithelium exhibited breakdown of actin filaments in the basal region of the cells within 18 hr of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. SDS-PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized muscle actin in polymerizing buffer led to an increase in actin monomer of approximately 25% in the UV-exposed solutions within 3–18 hr, whether assayed by ultracentrifugation or HPLC. The above indicates that elasmobranch lens epithelial cells contain UV-labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break down the actin structure in these cells. Furthermore, breakdown of purified polymerized muscle actin does occur due to near-UV light exposure. While there appears to be a direct effect of this UV radiation on the isolated actin, the lens damage would be modified by the cellular constituents and could be at the actin binding sites relative to supporting cell architecture.

Glucose metabolism in freshly isolated Müller glial cells from a mammalian retina.

Glucose metabolism was studied in isolated retinal Müller glial cells from the juvenile guinea pig. Cells, once enzymatically isolated and purified, were identified by morphological criteria, positive vimentin immunoreactivity, and histochemical staining for glycogen. Purified suspensions of Müller cells were obtained in quantities sufficient for biochemical analysis (approximately 2 x 10(5)/pair of retinas) and light microscopic autoradiography. In bicarbonate-buffered Ringer's medium containing 3H-2-deoxyglucose and no glucose, greater than or equal to 80% of the glucose analogue taken up intracellularly by Müller cells was phosphorylated to 3H-2-deoxyglucose-6-phosphate. In autoradiographs, this non-metabolized product provided visual evidence of glucose phosphorylation: the distribution of cell grains mirrored the morphology of individual Müller cells in situ. Exposure to the glycolytic inhibitor iodoacetate (500 microM) caused an 85% decrease in adenosine triphosphate (ATP) content; concomitantly, 3H-2-deoxyglucose-6-phosphate decreased by 90% and paralleled a dramatic decrease of cell labelling in autoradiographs, while levels of 3H-2-deoxyglucose did not change. In the continual absence of glucose, glycogen content decreased with time and this decrease was slowed by 36% in the presence of iodoacetate. This indicated that, in control conditions, glycosyl units from glycogen sustain cellular metabolism, and hence 3H-2-deoxyglucose phosphorylation. 3H-2-deoxyglucose-6-phosphate concentration was 43-fold less than that of ATP in the control conditions so that depletion of ATP during iodoacetic acid (IAA)-blocked glycolysis was not due to hexokinase activity. These results demonstrate that this preparation is adequate for quantitative studies of glucose metabolism at the cellular and molecular level in an important metabolic compartment of the mammalian retina.

Increased vasopressin immunoreactivity in the rat brain after a postmortem interval of 6 hours

Enhanced immunocytochemical staining of vasopressin-containing neurons was observed after incubation of rat brain slices in Ringer medium for 6 h at room temperature, as compared to brain tissue fixed immediately after death. Hypothalamic vasopressin neurons in the supraoptic nucleus, the paraventricular nucleus and the suprachiasmatic nucleus stained more intensely after a postmortem interval of 6 h. Extrahypothalamic vasopressin neurons (VP) in the bed nucleus of the stria terminalis, the medial amygdala and the locus coeruleus proved to be stained as well. Extrahypothalamic VP neurons in the locus coeruleus could, until now, only be visualized after in vivo pretreatment with colchicine. In addition, staining was observed at two new sites, the dorsal raphe nucleus and the lateral septum. Staining of VP was corroborated by application of different antibodies directed against the intact vassopressin molecules as well as by antibodies directed against the other parts of the vasopressin precursor molecule, i.e. neurophysin andglycopeptide. The specificity of the VP-staining was validated by using pre-immune serum and using Brattleboro rat brain tissue, resulting in a negative staining in both cases. Furthermore, homogenated punches of the suprachiasmatic nucleus were submitted to iso-electric focusssing on polyacrylamide gel, followed by press blotting and subsequent immunocytochemical staining for vasopressin. Iso-electric focussing enabled us to characterized and quantify peptides in the suprachiasmatic nucleus. The vasopressin content increased 6 h postmortem, while c-terminal glycopeptide and neurophysin levels remained stable. Similar results were observed in the suprachiasmatic nucleus from decapitated rats whose brains were left intact in the skull for 6 h at room temperature.

A comparison between fusion of rat hepatocytes induced by electric and by centrifugal fields

Fusion of hepatocytes suspended in Krebs—Ringer medium without calcium ions induced by electric fields is compared here to fusion induced during centrifugation. The results obtained demonstrate clearly the possibility of fusing hepatocytes in a weak centrifugal field without making use of an external transient electric field or any fusogenic component. This procedure seems more efficient and versatile than electrofusion; indeed, similar fusion yields can be obtained with a better cell viability. A hypothesis is presented which might account for the fusion results in a centrifugal field.

Development of a19F-n.m.r. method for studies on the invivoandin vitrometabolism of 2-fluoroaniline

1. A 19F-n.m.r. method has been developed for study of the metabolism of 2-fluoroaniline both after in vivo exposure of rats and in in vitro model systems. 2. From the 19F-n.m.r. spectrum of the 24 h urine it was calculated that over 90% of the dose was excreted within 24 h. The metabolic pattern showed that 85% of the metabolites were para-hydroxylated, 72% sulphated, 13% glucuronidated and 29% N-acetylated, 4-amino-3-fluorophenyl sulphate being the main urinary metabolite (53%). 3. In vitro studies of phase I metabolism of 2-fluoroaniline with rat liver microsomes was representative for the in vivo metabolism as hydroxylation in both systems was observed only at the para-position. 4. Phase I+II metabolism was studied in vitro in either isolated rat hepatocytes in suspension or in a 1 h recirculating liver perfusion system. In both these in vitro systems para-hydroxylation, N-acetylation, sulphation and glucuronidation of 2-fluoroaniline were observed. The ratio between glucuronidation and sulphation was dependent on sulphate availability. 5. Of the in vitro systems tested, hepatocytes in Krebs Ringer (sulphate limited) medium was the best model for in vivo metabolism. 6. The detection limit for fluoro-containing metabolites in this 19F-n.m.r. method was 1 MicroM for an overnight run using a Bruker CXP 300 spectrometer. From this it can be concluded that 19F-n.m.r. urine analysis is a useful tool in biomonitoring studies. For 2-fluoroaniline the method appears to be more sensitive than currently available h.p.l.c./t.l.c. methods. In addition, concentration of urine samples can result in either lower detection limits, or in shorter times needed for n.m.r. data acquisition. 7. N-acetylation is known to show genetic polymorphism. Therefore, the 19F-n.m.r. method, detecting all 2-fluoroaniline metabolites, has the additional advantage of eliminating the risk of obtaining false negatives for fast acetylators.

In vivo environmental temperature and the in vitro pattern of luminal acidification in turtle bladders. Evidence for HCO3 ion reabsorption.

In this study, it is shown how to transfer tared aliquots of (HCO3 + CO2)-containing luminal fluids directly into the mercury-sealed chamber of a modified Van Slyke apparatus and how to obtain direct as well as indirect manometric determinations of dissolved CO2 ([CO2]f) in each aliquot of such fluids. It is next shown that the pattern of in vitro luminal acidification in an isolated turtle bladder sac depends upon the prior in vivo ambient temperature to which the donor turtle had become adapted. Under in vivo conditions, the food intake, physical activity, and acid excretion of 32 degrees C-adapted turtles are greater than those of 21 degrees C or 26 degrees C-adapted turtles. Under in vitro conditions of incubating isolated bladder sacs (from 21, 26, and 32 degrees C turtles) in (HCO3 + CO2)-containing Ringer media at a single temperature (21 degrees C), the patterns of luminal acidification are as follows: (a) The rate of depletion of luminal [HCO3] is greatest in bladders from the 32 degrees C-adapted turtles. (b) Concomitant decreases in luminal [CO2]f, [HCO3], and pH (the 'CO2-decreasing patterns' of luminal acidification) develop in all bladders from 32 degrees C turtles, in half of those from 26 degrees C turtles, but in less than one-fifth of those from 21 degrees C-adapted turtles: and (c) a CO2-increasing pattern of luminal acidification is found in most of the bladders from 21 degrees C-adapted turtles. A postulated bicarbonate ion-reabsorbing pump is consistent with all of these patterns of luminal acidification.