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Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2'-deoxyuridine, 2'deoxyxanthosine, N(1)-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2'-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2'Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.

(R)-N-[4-[2-[[2-Hydroxy-2-(pyridin-3-yl)ethyl]amino]ethyl]phenyl]- 4-[4-(4-trifluoro-methylphenyl)thiazol-2-yl]benzenesulfonamide (1) is a potent and selective agonist of the human beta3-adrenergic receptor. We report herein the data from studies of the metabolism and excretion of 1 in rats. Five metabolites were identified in the bile of male Sprague-Dawley rats administered 3H-labeled 1 by either oral gavage (10 mg/kg) or intravenous injection (3 mg/kg). These included a pyridine N-oxide derivative (M2), a primary amine resulting from N-dealkylation and loss of the pyridinyl-2-hydroxyethyl group (M4), a carboxylic acid derived from N-dealkylation and loss of the pyridyl-2-hydroxyethyl amine (M5), and the corresponding taurine and isethionic acid conjugates (M1 and M3). Metabolites M1 and M3 also were identified in rats treated with M5 and were generated in incubations of M5 with rat liver subcellular fractions in the presence of ATP and coenzyme A with supplementary taurine or isethionic acid. These results suggest that M5 is the precursor of M1 and M3 and that the formation of these conjugated metabolites follows similar mechanisms of amino acid conjugation. On the other hand, M2, M4, and M5 were produced from 1 in an NADPH-dependent manner in incubations with liver microsomes from rats, dogs, monkeys, and humans. In human liver preparations, these routes of biotransformation were shown to be catalyzed by cytochrome P450 3A4. In a bidirectional transport assay, transport of 1 across a monolayer of cells expressing P-glycoprotein (Pgp) was observed to be similar to that of vinblastine, which is an established substrate of the transporter protein. This finding, together with the observation that the parent compound was excreted in the feces of bile duct-cannulated animals following intravenous dosing, suggests that 1 is subject to Pgp-mediated excretion from intestine of rats.

Many studies have suggested an antioxidant role for taurine, but few studies have directly measured its free radical scavenging activity. The aim of the present study was to directly determine the action of taurine and taurine analogs to inhibit peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR) to rhodamine. Taurine was also tested to determine if it could attenuate the toxicity of sodium nitroprusside (SNP) to neuronal cultures. Taurine at concentrations above 30 mM had a modest ability to inhibit peroxynitrite formation derived from SIN-1. Hypotaurine could inhibit peroxynitrite formation from both SIN-1 (decrease 75%) and SNP (decrease 50%) at 10 mM. Other taurine analogs (homotaurine, beta-alanine & isethionic acid) slightly potentiated DHR oxidation by SIN-1. Short-term (1-hour) treatment of PC12 cultures with either SNP (1-2mM) or taurine (20-40 mM) appeared to induce cellular proliferation. In contrast, 24-hour treatment with SNP (1 mM) induced cell death. Combination treatments with taurine and SNP appeared to interact in an additive fashion for both cell proliferation and neurotoxic actions. It appears unlikely that taurine is a major endogenous scavenger of peroxynitrite.

The aim of this study was to assess the relevance of chloride channels to the inhibitory effect of angiotensin II (ANGII) on renin secretion. We thus examined the effects of the chloride channels blockers IAA-94 and niflumic acid, the Na-K-Cl cotransport blocker bumetanide and substitution of isethionate for extracellular chloride on the action of ANGII on renin secretion from isolated perfused rat kidneys. Renin secretion prestimulated by isoproterenol (10 nmol/l) was almost completely blocked by ANGII with a concentration yielding a half-maximal response (EC50) of around 150 nmol/l. In the presence of IAA-94 and niflumic acid the EC50 for ANGII was shifted to about 400 nmol/l. In the presence of bumetanide and isethionate renin secretion responded more sensitively to ANGII and the EC50 for ANGII was below 100 nmol/l. On the assumption that the chloride equilibrium potential in renin-secreting cells is more positive than the membrane potential, our findings would suggest that the inhibitory effect of ANGII is enhanced when chloride entry is blocked and attenuated when chloride efflux is impaired. Activation of chloride channels therefore probably contributes to the inhibitory action of ANGII on renin secretion.

Kremer, E. and A. Lev-Tov. GABA-receptor–independent dorsal root afferents depolarization in the neonatal rat spinal cord. J. Neurophysiol. 79: 2581–2592, 1998. Dorsal root afferent depolarization and antidromic firing were studied in isolated spinal cords of neonatal rats. Spontaneous firing accompanied by occasional bursts could be recorded from most dorsal roots in the majority of the cords. The afferent bursts were enhanced after elevation of the extracellular potassium concentration ([K+]e) by 1–2 mM. More substantial afferent bursts were produced when the cords were isolated with intact brain stems. Rhythmic afferent bursts could be recorded from dorsal roots in some of the cords during motor rhythm induced by bath-applied serotonin and N-methyl-d-aspartate (NMDA). Bilaterally synchronous afferent bursts were produced in pairs of dorsal roots after replacing the NaCl in the perfusate with sodium-2-hydroxyethansulfonate or after application of the γ-aminobutyric acid-A (GABAA) receptor antagonist bicuculline with or without serotonin (5-HT) and NMDA. Antidromic afferent bursts also could be elicited under these conditions by stimulation of adjacent dorsal roots, ventrolateral funiculus axons, or ventral white commissural (VWC) fibers. The antidromic bursts were superimposed on prolonged dorsal root potentials (DRPs) and accompanied by a prolonged increase in intraspinal afferent excitability. Surgical manipulations of the cord revealed that afferent firing in the presence of bicuculline persisted in the hemicords after hemisection and still was observed after removal of their ventral horns. Cutting the VWC throughout its length did not perturb the bilateral synchronicity of the discharge. These findings suggest that the activity of dorsal horn neurons is sufficient to produce the discharge and that the bilateral synchronicity can be maintained by cross connectivity that is relayed from side to side dorsal to the VWC. Antagonists of GABAB, 5-HT2/5-HT1C, or glutamate metabotropic group II and III receptors could not abolish afferent depolarization in the presence of bicuculline. Depolarization comparable in amplitude to DRPs, could be produced in tetrodotoxin-treated cords by elevation of [K+]e to the levels reported to develop in the neonatal rat spinal cord in response to dorsal root stimulation. A mechanism involving potassium transients produced by neuronal activity therefore is suggested to be the major cause of the GABA-independent afferent depolarization reported in our study. Possible implications of potassium transients in the developing and the adult mammalian spinal cord are discussed.

To investigate the role of Cl- in the regulation of the basolateral transporters of salivary acinar cells, we have measured cell volume and intracellular pH (pHi) in perfused rat mandibular glands using proton NMR spectroscopy and BCECF fluorometry respectively. When perfusate Cl- was replaced by glucuronate, isethionate, methylsulphate, nitrate or thiocyanate, cell volume decreased slowly by about 15% over a 10-min period. Replacement with bromide, which substitutes for Cl- on the Na+-K+-2Cl- cotransporter, caused only a small (4%) reduction in cell volume. Replacement of Cl- by glucuronate, isethionate or methylsulphate evoked a biphasic increase in pHi consisting of a rapid initial increase followed by a slower secondary rise whose time course was similar to that of cell shrinkage. As judged by the effects of HCO3- omission, 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 1 mM amiloride, the initial rise in pHi was due to Cl-/HCO3- exchange while the secondary rise resulted from activation of Na+/H+ exchange. Although replacement of Cl- by nitrate or thiocyanate also caused cell shrinkage, these substituting anions were less effective in activating the exchanger. Therefore, while the upregulation of the exchanger following Cl- replacement may be due in part to cell shrinkage, there is also evidence for the involvement of an anion-sensitive regulatory mechanism. This would be consistent with the hypothesis that both changes in cell volume and in intracellular Cl- concentration contribute to the up-regulation of the exchanger following muscarinic stimulation.

The modulatory effects of taurine on [35S]-t-butylbicyclophosphorothionate (TBPS) binding to rat brain synaptic membranes were evaluated and compared with that of GABA. Taurine allosterically inhibited TBPS binding by interacting with a bicuculline-sensitive site, similar to GABA. Taurine was as effective as GABA but less potent. The potency of taurine inhibition of TBPS binding varied among brain regions with cerebellum > olfactory bulb > cortex, similar to that of GABA. Inhibition of TBPS binding to cortical membranes measured under nonequilibrium conditions yielded a dynamic biphasic inhibition curve that was similarly shaped for GABA and taurine. The effect of taurine on TBPS binding was pharmacologically specific in that beta-alanine and guanadinoethanesulfonate were as effective as taurine, while hypotaurine and alpha-aminoethylhydrogen sulfate were only partially effective at high concentrations, and isethionic acid was without effect. Taurine, similar to GABA, enhanced the effects of pentobarbital on TBPS binding when present at concentrations that were otherwise ineffective on their own. The results of these studies support the notion that taurine interacts with the GABA recognition site of the GABAA receptor complex.

The effects of changes in cell volume and pH on glycogen synthesis and glycolysis and their control by insulin were investigated in hepatocyte cultures. 1. Cell acidification, by increasing [CO2] from 2.5% to 5%, inhibited glycolysis and stimulated glycogen synthesis. The inhibition of glycolysis was also observed in Na(+)-free media and when K+ uptake was inhibited, but the stimulation of glycogen synthesis was abolished under these conditions, suggesting that it is secondary to ionic or volume changes. Alkalinization had converse effects on glycolysis and glycogen synthesis. 2. In HCO3(-)-containing media, replacement of NaCl with sodium acetate or potassium acetate, like acidification with CO2, inhibited glycolysis and stimulated glycogen synthesis. The latter correlated with an increase in cation content. Amiloride, an inhibitor of Na+/H+ exchange, inhibited both the increase in cation content and the stimulation of glycogen synthesis, suggesting that the latter is secondary to cell swelling. 3. Hypo-osmotic swelling increased glycogen synthesis in HCO3(-)-containing media, in both the absence and the presence of Na+ and at both 2.5% and 5% CO2, but it increased glycolysis in the presence of Na+ and at 2.5%, but not at 5%, CO2. In HCO3(-)-free media, during acidification and swelling, glycogen synthesis correlated with pH and not with cell volume, indicating that inhibition by acidification over-rides stimulation by swelling. 4. Stimulation of glycolysis by insulin was not additive with stimulation by alkalinization. The stimulation of glycogen synthesis by insulin was partially suppressed under alkaline conditions; it was markedly suppressed in isosmolar Na(+)-free media and restored by hypo-osmotic swelling. In hypo-osmolar Na(+)-free media insulin prevented the decrease in glycogen synthesis with decreasing [HCO3-], suggesting that it counteracts inhibition by acidification. 5. It is concluded that glycogen synthesis and glycolysis are both stimulated by cell swelling and inhibited by acidification, under certain conditions, but glycolysis is more sensitive to inhibition by acidification and glycogen synthesis to stimulation by swelling. Consequently, simultaneous swelling and acidification is associated with inhibition of glycolysis and stimulation of glycogen synthesis. Stimuli that cause swelling and alkalinization activate both glycogen synthesis and glycolysis, alkalinization being more important in control of glycolysis and swelling in control of glycogen synthesis. Both cell swelling and alkalinization are components of the mechanism by which insulin controls glycogen synthesis and glycolysis.

In rat hepatocytes grown on gas-permeable membranes (Petzinger et al. In Vitro Cell. Dev. Biol. 24: 491-499, 1988), cellular and canalicular potentials as well as input resistances were measured using two-channel microelectrodes. In HCO3(-)-containing solutions, we found -30.9 +/- 0.4 (SE) (n = 141) and -13.9 +/- 1.4 mV (n = 22) for cell and canalicular membrane potentials, respectively. There was no dependence of these parameters on the age of the primary culture. Canalicular input resistance, however, increased from 13.3 +/- 2.0 M omega (n = 4) at day 1 after seeding to 36.1 +/- 5.0 M omega (n = 9) at day 2 and stabilized thereafter, while cell input resistance continuously decreased from 37.0 +/- 3.3 M omega at 1 h (n = 6) to 5.2 +/- 2.1 M omega (n = 27) at 3 days after preparation. In ion substitution experiments there were no changes in the transference numbers for K+, Na+, or Cl- that could account for this effect. Cable analysis, however, revealed that the decrease in input resistance reflects a time-dependent increase in electrical coupling between cells. We conclude that rat liver cells on gas-permeable membranes are highly suited for the quantitative analysis of cell-to-cell interaction. In addition, cells and canaliculi are readily accessible with two-channel microelectrodes, making this preparation a promising tool for electrophysiological analysis of hepatocellular transport mechanisms.

Perfusion with high millimolar levels of taurine evoked a long-lasting potentiation (LLP-TAU) of synaptic transmission in the Schaffer-collateral CA1 region of the rat hippocampus. Although LLP-TAU showed some correlations to increases in the total taurine content of hippocampal slices, it could not be blocked by the taurine transport inhibitor guanidinoethanesulfonic acid (GES), which was able to significantly reduce total slice taurine uptake. Inhibition of GABA transport by either nipecotic acid or beta-guanidinopropionate failed to abolish LLP-TAU and had no significant effect on taurine uptake. The combination of GES and nipecotic acid also had no significant effect on LLP-TAU. Experiments with transportable structural analogs of taurine (beta-aminoisobutyric acid, homotaurine, and isethionic acid) suggest that activation of classical taurine transport pathways does not always yield a robust LLP-TAU. Hippocampal LLP-TAU could be significantly attenuated, however, by pre-incubation with submillimolar levels of taurine. In summary, the development of LLP-TAU in the rat hippocampus appears to be associated with the intracellular accumulation rather than the activation of known transporters of taurine, but the precise means of its accumulation remains to be identified.

Taurine has been shown to be an effective scavenger of hypochlorous acid (HOCl). The role of HOCl is well established in tissue damage associated with reperfusion injury mediated by neutrophils. The role of HOCl in CNS injury and inflammatory reactions has not been well established. Myeloperoxidase activity is present in the CNS and it has been associated with ischemic injury. The aim of the present study was to determine the cytotoxicity of HOCl in a neuronal cell line (PC12) and the ability of taurine to prevent or reverse neurotoxicity. PC12 cells were grown in 96 well plates at a plating density of approximately 100,000 cells per well. HOCl was made up fresh from NaOCl for each experiment and the concentration verified spectrophotometrically. PC12 cells were exposed to HOCl for 1 hour in phosphate-buffered saline. Taurine was added at the time of HOCl treatment and in some experiments a post-treatment with taurine was performed by adding 1 or 10 mM taurine to the culture media (RPMI 1640). The cells were allowed 24 hours to recover and viability was determined using a tetrazolium-based (MTT) assay. The first series of experiments evaluated the toxicity of HOCl and the efficacy of taurine to protect PC12 cells. HOCl at 50 microM reduced PC12 cell viability by 50% and 150 microM reduced viability to25% of control levels. Taurine (0.5-20 mM) was tested for cytoprotection against 150 microM HOCl and PC12 cells treated with 0.5 mM taurine exhibited only a 20% reduction in viability compared to untreated controls. Taurine concentrations of 1 mM or higher provided nearly 100% protection against HOCl. A second study was performed comparing taurine to beta-alanine, glutathione and isethionic acid. HOCl (100 microM) reduced viability to 25 +/- 1% of controls and taurine, beta-alanine and glutathione at 1 mM provided nearly complete protection. In contrast, isethionic acid, which lacks an amino group, failed to provide protection. Taurine (1 or 10 mM) added after 50 microM HOCl treatment did not provide any protection and PC12 cell viability was reduced to39% of controls. In contrast, if taurine (50 microM) was present during the HOCl treatment and 1 mM taurine was added after the treatment, PC12 cell viability was 80 +/- 5% of controls. A combination of 250 microM taurine during the HOCl treatment and 1 mM taurine post-treatment produced 100% protection. These results clearly show that taurine is an efficient scavenger of HOCl and can prevent neuronal damage caused by HOCl. Since myeloperoxidase expression in the CNS is increased by ischemia, one function of taurine released during an ischemic event may be to scavenge HOCl and provide neuroprotection.

Mass spectrometry-based metabolomics has previously demonstrated utility for identifying biomarkers of ionizing radiation exposure in cellular, mouse and rat in vivo radiation models. To provide a valuable link from small laboratory rodents to humans, γ-radiation-induced urinary biomarkers were investigated using a nonhuman primate total-body-irradiation model. Mass spectrometry-based metabolomics approaches were applied to determine whether biomarkers could be identified, as well as the previously discovered rodent biomarkers of γ radiation. Ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry analysis was carried out on a time course of clean-catch urine samples collected from nonhuman primates (n = 6 per cohort) exposed to sham, 1.0, 3.5, 6.5 or 8.5 Gy doses of (60)Co γ ray (∼0.55 Gy/min) ionizing radiation. By multivariate data analysis, 13 biomarkers of radiation were discovered: N-acetyltaurine, isethionic acid, taurine, xanthine, hypoxanthine, uric acid, creatine, creatinine, tyrosol sulfate, 3-hydroxytyrosol sulfate, tyramine sulfate, N-acetylserotonin sulfate, and adipic acid. N-Acetyltaurine, isethionic acid, and taurine had previously been identified in rats, and taurine and xanthine in mice after ionizing radiation exposure. Mass spectrometry-based metabolomics has thus successfully revealed and verified urinary biomarkers of ionizing radiation exposure in the nonhuman primate for the first time, which indicates possible mechanisms for ionizing radiation injury.

We investigated the relevance of anions for the regulation of renin secretion from the kidneys. For this purpose we measured renin release from isolated rat kidneys that were perfused with medium containing either 120 mmol/l (normal) chloride or 95 mmol/l of isethionate, acetate, or nitrate anions in exchange for equimolar amounts of chloride. Lowering the extracellular chloride concentration by either of these maneuvers significantly enhanced renin secretion rates (RSR) at a perfusion pressure of 100 mmHg. Increasing pressure above 100 mmHg inhibited renin release in the presence of isethionate and acetate but not with nitrate anions. The renin stimulatory effects of isethionate and acetate but not that of nitrate anions disappeared in the presence of bumetanide (100 mumol/l), an inhibitor of macula densa chloride transport. Activation of renin secretion by isethionate and acetate was blunted with 100 pmol/l angiotensin II (ANG II), whereas tenfold higher concentrations of ANG II were required to attenuate the effect of nitrate ions. The amount of renin released in the presence of nitrate was fully additive to RSR values obtained with maximally effective doses of isoproterenol. These findings are consistent with the idea that impermeant anions such as isethionate and acetate enhance renin secretion from the kidneys predominantly via the tubular macula densa mechanism. The stimulatory influence of membrane-permeable nitrate anions appears to involve additional pathways and is mediated by a decreased calcium sensitivity of the renin secretory process rather than resulting from an adenosine 3',5'-cyclic monophosphate-dependent action.

Brain lipids were labelled with [1-14C]-isethionyl acetimidate and purified by sequential thin layer chromatography. Four labelled peaks were obtained, the first ones migrating with the same Rf as glycosyl-phosphatidylinositol (GPI). Further proof of the isolation of GPI was obtained by the observations that 44.8% of the radioactivity associated with the lipid in peak I was converted to the water phase by the effect of a PI-specific phospholipase C, and that the soluble material so obtained produced a dose-dependent inhibition of cAMP-dependent protein kinase activity. These findings indicate a biological equivalence between GPI and its polar head group from rat brain and those described in other cell types, and are consistent with the proposed role of these molecules in cellular signalling.

The sensitivity of renin release to changes in anion and calcium concentrations was assessed in superfused rat glomeruli with attached juxtaglomerular cells. Isosmotic substitution of Cl-with gluconate (1/12, 1/6, 1/3, 2/3, or total exchange), isethionate (15 or 101 mM), or sulfate (10 mM) inhibited renin release reversibly. Substitution of Cl- with nitrate (101 mM) stimulated renin secretion. Substitution with iodide (15 or 101 mM) had no consistent effect. The stimulation induced by calcium-free solutions was high in May and low in September. In the absence of chloride, the response to calcium-free solution was inhibited similarly all year. In May reintroduction of calcium and chloride stimulated renin release, suggesting that releasable renin had been stockpiled during the exposure to calcium-free solution. In September reintroduction of calcium and chloride inhibited renin release. It is concluded that the renin secretory process has a demand for permeant anions. The stimulation caused by low external calcium involves at least two mechanisms: one is anion sensitive, powerful, varies with the season, and includes a recruitment phenomenon; another is anion insensitive and weak.

We have investigated the topography of a glycosyl-phosphatidylinositol implicated in insulin action by a combination of two complementary methods: (a) chemical labelling with a non-permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with beta-galactosidase (EC 3.2.1.23) or phosphatidylinositol-specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl-phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl-phosphatidylinositol to the exterior. Upon insulin addition (10 nM), about 60% of the total glycosyl-phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with beta-galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosyl-phosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol-specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl-phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulin-sensitive cells.

Transport of taurine has been demonstrated in capillary preparations from adult rat brains using [3H]taurine. Taurine transport is mediated by a saturable high-affinity system which is entirely dependent on sodium ions. The apparent maximal influx (Vmax) and half-saturation concentration (Km) corresponded to 1.06.10(-4) mumol/min per mg protein and 27.5 microM, respectively. Competition experiments in the presence of sodium ion showed that [3H]taurine uptake was strongly inhibited by 0.1 mM unlabeled structural analogues of taurine such as beta-alanine and hypotaurine as well as unlabeled taurine. gamma-Aminobutyric acid (GABA) (0.1 mM) inhibited the uptake of labeled taurine by 30%, whereas isethionic acid, L-methionine, L-2,4-diaminobutyric acid, glycine, L-cysteinesulfonic acid and cystamine did not exhibit any inhibitory effect. The results suggest that the Na+ gradient is the principal source of energy for taurine transport into isolated brain capillaries. This transport system may play an active role in the regulation of taurine concentration in the brain extracellular space.

The effects of taurine on ATP-dependent calcium ion uptake and protein phosphorylation of rat retinal membrane preparations were investigated. Taurine (20 mM) stimulates ATP-dependent calcium ion uptake by twofold in crude retinal homogenates. In contrast, it inhibits the phosphorylation of specific membrane proteins as shown by acrylamide gel electrophoresis and autoradiography. The close structural analogue of taurine, 2-aminoethylhydrogen sulfate, demonstrates similar effects in both systems, i.e., stimulation of ATP-dependent calcium ion uptake and inhibition of protein phosphorylation, whereas isethionic acid and guanidinoethanesulfonate have no effect on either system. A P1 subcellular fraction of the retinal membrane preparation that contains photoreceptor cell synaptosomes has a higher specific activity for the uptake of calcium ions. Phosphorylation of specific proteins in the P1 fraction is also inhibited by the addition of 20 mM taurine. Taurine has no effect on retinal ATPase activities or on phosphatase activity, thus suggesting that it directly affects a kinase system.

— Glucose oxidation by rat brain cortex slices is inhibited by low concentrations of triethyltin and this sensitive inhibition is shown to be chloride-dependent. Pyruvate oxidation is only inhibited at high concentrations of triethyltin whether chloride is present or not. In contrast, the stimulation of both glucose and pyruvate oxidation observed with low concentrations of triethyltin prior to inhibition is chloride-dependent. The results are discussed in relation to the chloride-hydroxide exchange reaction known to be mediated across the inner mitochondrial membrane by triethyltin and other organo-metals.

Two sets of taurine receptors on rat heart sarcolemma have been identified. The high affinity taurine receptors (Kd=3.5×10−4M) show a non-cooperative binding profile while the low affinity taurine receptors exhibit positive cooperativity. Taurine binding to the membrane exhibits a typical bell shaped pH profile with maximum binding occurring at pH 8.0. The maximum temperature for binding is 24°C. The effect of various taurine analogues on the receptors was investigated. It was found that binding is prevented by hypotaurine and inhibited to a lesser degree by isethionic acid and cysteine sulfinic acid, while β-alanine was found to increase taurine binding. The effect of several hydrolytic enzymes was also examined and it was shown that several proteases and phospholipase C inhibit binding. The results indicate that the taurine receptors are membrane bound proteins in a phospholipid environment.

We report that mammalian tissues posses hypotaurine (2-aminoethane- sulfinate) aminotransferase activity. One product of transamination, sulfinoacetaldehyde could theoretically undergo internal oxidation-reduction leading to isethionate (2-hydroxyethanesulfonate). This hypothesis was examined. No isethionate was formed in vitro .

Effects of taurine and related compounds on Ca-uptake by the disc membranes prepared from dark-adapted frog retina were studied. Taurine stimulated ATP-dependent Ca-uptake and the turnover of 45Ca in the disc membranes without affecting basal activity, but it was not observed with the synaptic plasma membranes from rat brain. The stimulatory effect appears to be specific to taurine, since cysteine sulfinic acid, hypotaurine, isethionic acid, β-alanine and γ-aminobutyric acid (GABA) did not stimulate Ca-uptake. The maximal activation, observed at about 30 mM taurine, was about 3 fold, and the Km value for taurine was 10 mM. These results might suggest that taurine modifies translocation of Ca ion in the rod outer segment.

The biosynthesis of isethionate from taurine in mammalian tissue has been reexamined. In vivo metabolism of taurine to isethionate was demonstrated but it was shown that a number of acyltaurine metabolites also behave like isethionate on the conventional dual column ion-exchange chromatographic analytical system. Hydrolysis of these column effluates coupled with high-voltage electrophoresis resolves this ambiguity. In vivo formation of isethionate from taurine in mammals seems to occur from gut microorganism metabolism since: (a) germ-free mice could not convert taurine to isethionate, (b) gut anaerobes were able to metabolize taurine, (c) in vitro rat and mouse tissue failed to metabolize taurine to isethionate. These findings are in conflict with earlier reports.

Somatostatin receptors appear to be localized to secretory granules in pancreatic islet homogenates. Recruitment of these receptors to the islet-cell surfaces may mark the contact event between secretory granules and plasma membranes before release of insulin by fission. Isethionate, an impermeant anionic replacement for chloride, blocks the release step but does not affect receptor recruitment. By contrast, low concentrations of phenothiazine drugs, such as trifluoperazine and promethazine, inhibit both receptor recruitment and secretion. Scatchard analysis of phenothiazine effects on somatostatin receptors reveals that these drugs reduce the number of receptors but do not affect the affinity of the receptor for somatostatin. These data indicate that membrane contact and fission steps during exocytosis can be biochemically separated.

The rates of disappearance of tritiated isethionic acid (2-hydroxyethanesulfonic acid) in eight regions of the rat central nervous system were studied. By utilizing the technique of graphical analysis ("curve peeling"), it was determined that seven areas (striatum, diencephalon, pons-medulla, midbrain, hippocampus, spinal cord, and cortex) exhibited triphasic (fast, intermediate, and slow) disappearance rates while only the cerebellum displayed a biphasic (fast, slow) rate. The half-lives for the fast component in the different regions of the central nervous system varied from 0.5 hr (midbrain and cortex) to 1.5 hr (diencephalon, cerebellum, and spinal cord): the half-lives for the intermediate component of the triphasic rate varied from 3.5 hr in the midbrain and spinal cord to 5.5 hr in the hippocampus. Half-lives estimated for the slow component of the multiphasic rate of disappearance of tritiated isethionic acid ([3H]ISA) varied from 28 hr (cerebellum) to 90 hr (spinal cord).

d -Serine and d -2,3-diaminopropionic acid ( d -DAPA) induce necrosis of the epithelial cells of the proximal straight tubules of the rat kidney. Nineteen compounds with chemical structures and/or functional groups similar to d -serine or d -DAPA were tested for their protective effect against the induction of this injury. The acute tubular necrosis inducible by d -serine and d -DAPA was prevented by d -alanine, d -threonine, d -homoserine, dl -α-methyl serine, β-hydroxy- dl -leucine, and α-aminoisobutyric acid. In addition, protection from necrosis due to d -DAPA, but not that due to d -serine, was found with l -threonine, l -serine, l -2,3-diaminopropionic acid, and 3-hydroxypropionic acid. Little or no protective effect against d -serine of d -DAPA was afforded by l -alanine, β-alanine, dl -isoserine, dl -3-hydroxybutyric acid, l- or dl -lactic acid, 2-amino-2-methyl-1-propanol, taurine, and isethionic acid. The compounds that protected against d -serine-induced necrosis had in common the d -configuration (except for aminoisobutyric acid), the α-amino group, and the carboxyl group. The structural characteristics and chirality of the protective agents against d -DAPA were more varied and less definable than for d -serine. d -Alanine, dl -α-methyl serine, and α-aminoisobutyric acid protected on a mole-for-mole basis against d -serine-induced necrosis. d -Alanine was protective against d -serine even when given 30 min after d -serine administration. These results suggest that protection may be due to competition with d -serine or d -DAPA for a cellular or transport binding site.

The effects of stimulating the parasympathetic innervation to rat submaxillary gland on ductal transport of Na, K, water, and transepithelial PD, were tested in the main excretory duct during perfusion through its lumen. During nerve stimulation, transepithelial PD was consistently decreased, usually by about 15 mV, and this decrease could be blocked by atropine but not by adrenergic blocking agents. Net flux of Na, K, or water was not significantly changed during stimulation. One-way flux of Na from the lumen also was unaffected. The decrease in PD during stimulation was not affected by substituting isethionate for Cl or increasing [K] in the luminal perfusion medium. It is concluded that parasympathetic effects are exerted on the ductal cells, but that these probably do not involve conductance changes at the luminal membrane.

Changes in cell volume and alanine, Na+ and K+ concentrations and fluxes were studied in rat isolated hepatocytes or perfused liver. Addition of 10 mM alanine elicited a noticeable entry of Na+ which accumulated into the cells, along with a leakage of cell K+. Concurrently, the cell volume was 13% increased. These processes were also present with perfused livers in which alanine uptake, Na+ entry and cell swelling were proportional to the induction of the A system. K+ efflux was parallel to cell swelling and occurred very early after exposure of liver cells to alanine (and was transitory). The rôle of extracellular anions was investigated: alanine transport was not affected by replacement of Cl- and HCO-3 by a permeant anion whereas it was inhibited with the non-permeant anion isethionate. The present results indicate that the disturbances of the ionic gradients consecutive to alanine transport in isolated hepatocytes also operate in the liver organ itself. The possible implications of these phenomenons are discussed.

1. A phosphorus-fluorine splitting enzyme (DFPase) from squid nerve hydrolyzes DFP 5–10 times faster than it hydrolyzes another P-F compound, Soman, whereas a superficially similar enzyme from rat kidney hydrolyzes Soman 20–40 times faster than it hydrolyzes DFP, all under comparable conditions. 2. The DFPase from rat kidney is stimulated 2- to 3-fold by 4 × 10−4 M Mn2+, whereas the DFPase from squid nerve is unaffected or slightly inhibited by 4 × 10−4 M Mn2+. 3. These observations form the basis for distinguishing between a squid type DFPase and a mammalian DFPase, the names not being rigorously indicative of enzyme source or substrate. 4. When these criteria are applied to a P-F splitting enzyme found in squid saliva, the enzyme is identifiable as squid type DFPase. There is a significantly higher level of this enzyme in whole saliva from female squids than in whole saliva from male squids. This squid type DFPase is different from the proteinous toxin also found in squid saliva.

A phosphatidylinositol-glycan has been implicated in the mechanism of action of insulin. Some of the actions of insulin may be mediated by the generation of the polar head group of this phosphatidylinositol-glycan. Localisation of the insulin-sensitive phosphatidylinositol-glycan was investigated by reacting the glycophospholipid with the imidoester [1-14C]-isethionyl acetimidate. The present results indicate that most of the insulin-sensitive phosphatidylinositol-glycan is localized at the plasma membrane of rat liver, being 85% of the glycophospholipid present at the outer surface of the cell. These results suggest a paracrine action of insulin.

Sulfur of methionine is shown to flow into cysteine via the transsulfuration pathway and ultimately into taurine and sulfate via hypotaurine. Labelled intermediates, homocysteine and cystathionine, were detected in rat brain slices after the incubation with [35]methionine. [35S]Cysteine was identified as cystine after addition of carrier and crystallization. Cysteine sulfur is shown to flow into taurine and sulfate via cysteine sulfinate and hypotaurine. The identification of hypotaurine gives strong support to the proposed pathway of taurine formation. Taurine is shown to undergo reactions to yield isethionic acid in very small amounts. The mechanism of the conversion has not been worked out but proof is furnished that the conversion is enzymatic.

Structural analogues of taurine that are inhibitors of the rat liver microsomal synthesis of taurocholic acid are reported. Isethionic acid, the hydroxy analogue of taurine, is the most potent inhibitor ( I 50 = 110 μM ). Aminomethanesulfonic acid, a taurine analogue which has a single carbon bridge between the amino and sulfonic acid moieties, is also a very potent compound ( I 50 = 688 μM ). Hydroxypropanesulfonic acid ( I 50 = 3125 μM ), the higher homologue of isethionic acid, and aminoethane-sulfuric acid ( I 50 = 3750 μM ) are considerably less potent inhibitors. Hypotaurine I 50 = 7500 μM ) and glycine ( I 50 = 8800 μM ), both substrates for the enzyme system, decrease taurocholate formation but only when used at extremely high concentrations.

Summary Potassium α-hydroxyethane [ 35 S]sulphonate was administered in the drinking-water to male albino rats (weighing 100 g) as a single dose equivalent to 50 mg SO 2 /kg. After 48 hr, 72–85% of the 35 S had been excreted in the urine and 10–15% in the faeces. After 2 wk, less than 1.5% remained in the carcass. Of the urinary 35 S, 90–95% was in the form of inorganic sulphate and the rest was not precipitable by barium. No evidence was obtained that unchanged α-hydroxyethane sulphonate was excreted. No signs of toxicity were observed.

The influx of [14C]isethionate (ISA) into rat brain slices was studied with and without taurine. This influx was relatively rapid, but took place largely by a non-saturable, passive mechanism, which transferred much less ISA into the brain cells than taurine. Taurine inhibited the influx of ISA competitively (Km=50 and 100 μmol/l) at low ISA concentrations, and ISA that of taurine non-competitively (V=200 and 400–700 μmol×min−1×kg−1 wet weight) at high taurine concentrations. It thus appears that ISA and taurine may have a small number of common transport sites at brain cell membranes, but these are apparently of little significance for the total transport of ISA.

1. Each of five triorganotin and five triorganolead compounds was shown to perturb mithochondrial functions in three different ways. One is dependent and two are independent of Cl- in the medium. 2. Structure-activity relationships for the three interactions are described, and compounds suitable as tools for the separate study of each process are defined. 3. In a Cl- -containing medium trimethyltin, triethyltin, trimethyl-lead, triethyl-lead and tri-n-propyl-lead all produce the same maximum rate of ATP hydrolysis and O2 uptake; this rate is much less than that produced by uncoupling agents such as 2,4-dinitrophenol. 4. Increase in ATP hydrolysis and O2 uptake are measures on energy ultilization when triogranotin and triorganolead compounds bring about an exchange of external C1- for intramitochondrial OH- ions. Possible rate-limiting steps in this process are discussed. 5. In a C1- -containing medium ATP synthesis linked to the oxidation of beta-hydroxybutyrate or reduced cytochrone c is less inhibited by triethyltin or triethyl-lead than is ATP synthesis linked to the oxidation of succinate, pyruvate or L-glutamate. 6. The inhibition of ATP synthesis linked to the oxidation of both beta-hydroxybutyrate and reduced cytochrome c consists of two processes: one is a limited uncoupling and is C1- -dependent and the other is a C1- -independent inhibition of the energy-conservation system. 7. The different sensitivities to inhibition by triethyltin of mitochondrial functions involving the oxidation of beta-hydroxybutyrate and succinate are compared and discussed.

Release of prolactin from both normal pituitary cells and rat pituitary tumor (GH) cells is an osmotic process that is dependent upon chloride. The long term growth rate of GH-cells in medium in which chloride was exchanged with isethionate was completely normal, but, by 48 h, isethionate substitution resulted in a 70% decrease in the concentration of internal and secreted prolactin. Isethionate caused a much smaller reduction in growth hormone production (less than 20%). These results suggest that exchange of chloride with isethionate is inhibiting the synthesis of prolactin. Reduction of intracellular levels of prolactin in cells grown in isethionate-containing medium was evident by 30 h, and the level of prolactin was reduced 92% at 96 h. This reduction in the internal concentrations of prolactin was reversed when the cells were returned to normal medium containing chloride with a t1/2 of 48 h. Addition of epidermal growth factor and the calcium channel agonist BAY K 8644 to cells in medium containing chloride increased internal prolactin by 400%, and isethionate exchange reduced the response by 85%. To confirm that isethionate exchange was inhibiting the synthesis of prolactin, mRNA concentrations for prolactin and actin were determined. Both basal and hormone-stimulated levels of prolactin mRNA were reduced 70 to 90% by isethionate exchange, while actin mRNA levels did not change. To determine whether the effect of isethionate was at the level of gene transcription, GH-cells were transfected with a prolactin-chloramphenicol acetyltransferase fusion gene and chloramphenicol acetyltransferase expression was assessed using cells in chloride and isethionate-containing media. Both basal and hormone-stimulated synthesis of chloramphenicol acetyltransferase driven by the prolactin promoter was inhibited by isethionate exchange. These studies demonstrate that exchange of medium chloride with isethionate inhibits the synthesis of prolactin at the level of transcription.

The ‘chemiosmotic’ model for secretion proposed by Pollard and his colleagues (Int. Rev. Cytol. 58, 159–197, 1979) was tested with pheochromocytoma cells. Contrary to the prediction of this model, norepinephrine secretion did not require the presence of a permeant anion in the medium. Secretion was not blocked by replacing much of the Cl − of the medium with isethionate or by replacing all of the Cl − salts of the medium with isotonic sucrose. Biochemical evidence is presented to indicate that the cells secreted by the normal exocytotic mechanism in the sucrose medium. Making the normal bathing medium hypertonic with 300 mM sucrose increased the basal level of norepinephrine release, but alos suppressed secretion in response to a strong secretagogue (1 mM Ba 2+ ). The data indicate that the Pollard model does not apply to pheochromocytoma cells, but suggest the possible involvement of osmotic pressure in exocytosis.

1. Responses of single ganglion cells in the isolated rat superior cervical ganglion to gamma-aminobutyric acid (GABA) applied via the bathing medium were recorded using intracellular micro-electrodes. 2. GABA produced a large fall in cell input resistance, frequently to immeasurable levels. In thirteen cells showing a modest response to 100 muM GABA, input resistance fell from 50-5 +/-9-5 to 15.9 +/- 3-2 Momega (means +/- S.E. of mean). After correction for resistance leaks introduced by the impaling electrode, the resting membrane resistance Rm and the resistance of the GABA-shunt Rg in these cells were calculated to be 79-3 +/- 16-6 and 35-0 +/- 9-5 Momega respectively. 3. Cells with recorded resting membrane potentials greater than -42 mV were depolarized by GABA; at resting potential less than -42 mV they were hyperpolarized...

Acinar secretion was studied in the caerulein-stimulated perfused rat pancreas. Unstimulated glands secreted at a mean basal rate of 1.1 microliters X g-1 X min-1 (SD = 0.74), which was not altered by perfusate anion substitution or by transport-blocking drugs. Caerulein evoked a maximum response (6.8 microliter X g-1 X min-1, SE = 0.36, n = 8) at a concentration of 18 pmol/l. Replacement of perfusate bicarbonate with either chloride or acetate did not significantly alter the stimulated secretory rate. In contrast, replacement with acetate of either chloride alone or chloride and bicarbonate reduced the rate of stimulated secretion by 75-80%, and replacement with isethionate abolished the response altogether. In glands perfused with solutions containing chloride but not bicarbonate, furosemide (10(-5) M), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 10(-4) M), amiloride (10(-4) M), and methazolamide (10(-4) M) all reduced the secretory response by more than 70%. When bicarbonate was included in the perfusate, the inhibitory effects for the same doses of blockers were much less, and except for methazolamide the same level of maximum inhibition was obtained when the blocker doses were increased 10-fold. The results suggest that rat pancreatic acini utilize a different secretory mechanism from the sodium chloride-carrying symport postulated to be most important in rat mandibular glands. The simplest model to explain our results would involve paired, basolateral antiports for Na-H and Cl-HCO3 exchange. We cannot exclude the presence of a Na-Cl symport, but if present, its role appears to be minor.

It has been concluded that tubulo-glomerular feedback mechanism is triggered by changes in NaCl concentration ([NaCl]) at the macula densa. This conclusion is based on the demonstration that changes in filtration rate produced during retrograde perfusion of the loop of Henle depend upon the perfusate [NaCl]. Experiments were performed to evaluate whether the effect on glomerular function of orthograde perfusion of the loop of Henle is consistent with this conclusion. Early proximal flow rate ( $$\dot V_{EP} $$ ), stop-flow pressure (P SF), early distal chloride concentration ([Cl]), and flow rate were measured during perfusion of the loop of Henle with mannitol solution (300 mosm kg−1), 30 mM NaCl+mannitol (300 mosm kg−1), 140 mM Na isethionate and artificial tubular fluid. When distal flow exceeded 10 nl min−1, the magnitude of the glomerular response was predictable from the [Cl]. The linear regression line, $$\Delta \dot V_{EP} = - 0.027{\text{ }}[Cl]{\text{ + 4}}{\text{.3}}$$ , did not differ from that obtained previously with the retrograde technique. Retrograde perfusion with 140 mM Na isethionate was without effect on $$\dot V_{EP} $$ . We conclude that the effect on glomerular function of perfusion of the loop of Henle in either an orthograde or a retrograde direction with these solutions depends upon the chloride concentration at the macula densa.

Evidence is presented that preparations of rat liver microsomes are capable of conjugating isethionic acid with cholic acid. Thin-layer chromatography experiments indicate that this newly described conjugated bile acid migrates with the same R f value as taurocholic acid, a structurally similar analogue. Dilution experiments utilizing both labeled cholic acid (tritium) and isethionic acid (sulfur-35) demonstrate the incorporation of both isotopes into a cholyl-conjugate of isethionic acid. Acid hydrolysis of enzymatically synthesized cholyl[ 35 ]isethionic acid and subsequent thin-layer chromatography of the degradation products also indicate that isethionic acid is conjugated with cholic acid. Formation of the cholyhydrazide derivative of cholyl-isethionic acid confirms an ester linkage between the carboxyl moiety of cholic acid and the hydroxyl moiety of isethionic acid. Rats receiving radioactive isethionic acid by stomach tube synthesized approximately 28 nmoles cholyl-isethionic acid compared with 980 nmoles taurocholic acid when radioactive taurine was administered under similar conditions.

A gas-liquid chromatographic assay has been developed to measure isethionic acid after methylation with diazomethane. The identity of the products of methylation has been confirmed by mass-spectrometry and nuclear magnetic resonance spectroscopy. The method was used to measure isethionic acid in rat heart, dog heart and rat brain. The assay was validated by measuring isethionic acid on squid axoplasm. We have been able to detect only trace amounts of isethionic acid in rat brain (0.2 mg/100g) and rat heart (0.1 mg/ 100g). None was found in dog heart.

Myocardial taurine level was found to be greater in the rat than in the guinea-pig. Taurine inhibited the development of arrhythmias caused by the i.v. infusion of ouabain in the rat. When ouabain induced arrhythmias, the myocardial taurine level decreased. Both effects were blocked by pretreatment of propranolol. The uptake of taurine into the heart did not necessarily result in the reversal of the PR interval prolonged by ouabain. The protecting effect of taurine against ouabain toxicity was not dependent upon the change in myocardial total ATPase activity.

Granule fusion and the subsequent fission leading to hormone discharge are distinct and separable events in exocytosis. As an index of fusion, we followed the recruitment of granule-bound somatostatin receptors to the islet surface, an event which accompanies secretion vesicle migration and insulin secretion. Granule fission was monitored by measuring insulin release. Substitution of the impermeant salt sodium isethionate for NaCl led to a 90% decrease in glucose-stimulated insulin release with no inhibition of somatostatin receptor recruitment. The phenothiazine drugs, trifluoperazine and promethazine, believed to inhibit calcium-sensitive proteins involved with stimulus-secretion coupling blocked insulin release and somatostatin receptor recruitment in parallel. This suggests that these agents suppress intracellular events promoting fusion of the secretion granule with the plasma membrane. IBMX appears to stimulate specifically granule fission since IBMX-induced insulin release occurs acutely without an increase in somatostatin receptor recruitment. Sodium isethionate, which inhibits granule lysis, blocked IBMX-stimulated insulin release.

Replacement of extracellular chloride with isethionate or methylsulphate causes an increased efflux of 1-[14C]-GABA from the in vivo superfused rat cuneate nucleus. This raises the question of the suitability of these anions as inert substitutes for chloride in studies on the ionic dependency of membrane phenomena in the central nervous system.