Inhibition of Glutamate-Induced Intensification of Free Radical Reactions by Gangliosides: Possible Role in Their Protective Effect in Rat Cerebellar Granule Cells and Brain Synaptosomes

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (I00 M) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na⁺, K⁺-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10–100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.     

Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes

The association of [³H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25°C follows a sigmoid path with a Hill coefficient h=1.25±0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33±0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5±7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)–1]^–1. Arthenius-type plots of [(r_o/r)–1]^–1 indicated that the lipid separation of the synaptosomal membranes at 23.4±1.2°C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8±0.8°C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10^–10–10^–4 M). These effects were reversed by naloxone (10^–4 M) which alone was ineffective in changing NO and cGMP production. We propose that Met-enkephalin achieved these effects through receptor mediated perturbations of membrane lipid structure and that inhibition of the L-Arg/NO/cGMP pathway in the brain may result in the antinociceptive effects of Met-enkephalin.     

The effect of boromycin on the Ca2+ homeostasis

A boron-containing antibiotic, boromycin (BM), was found to influence the Ca²⁺ homeostasis in both excitable and non-excitable cells. In non-excitable cells (human erythrocytes and leucocytes) it inhibited the resting passive⁴⁵Ca²⁺ transport in 10^–6–10^–5 mol/L concentrations. In human erythrocytes, the passive ⁴⁵Ca²⁺ transport induced by the presence of 1 mmol/L NaVO₃ was inhibited by boromycin (90% inhibition) as well. The inhibitory effect of BM on the NaVO₃-induced passive ⁴⁵Ca²⁺ transport was diminished in the presence of inhibitory concentrations of nifedipine (10 mol/L – 60% inhibition) or of those of K⁺ _o (75 mmol/L – 20% inhibition). On the other hand, in rat brain synaptosomes, and rat cardiomyocytes, BM stimulated the passive ⁴⁵Ca²⁺ transport in resting cells at similar concentrations. In rat cardiomyocytes the stimulation was transient. The stimulatory effect on the passive ⁴⁵Ca²⁺ transport in rat brain synaptosomes was accompanied with the increase of cytoplasmic Ca²⁺ concentration measured by means of the entrapped fluorescent Ca²⁺ chelator fura-2. The stimulatory effect of BM was diminished when synaptosomes were pre-treated with veratridine (10 mol/L) which itself stimulated the passive ⁴⁵Ca²⁺ transport. At saturating concentrations of veratridine, no stimulatory effect of BM was observed. These results could be explained by the indirect interaction of BM with both Ca²⁺ and Na⁺ transport systems via transmembrane ionic gradients of monovalent cations and could be useful in determining whether the cells belong to excitable, or non-excitable cells.     

Oxidant Production by Human B Lymphocytes: Detection of Activity and Identification of Components Involved

The O⁻₂-generating NADPH oxidase, originally thought to be expressed only in phagocytic cells of the immune system, can also be expressed in B lymphocytes. Epstein–Barr virus-transformed B cell lines (EBV-BL) can generate O⁻₂at rates corresponding to between 1 and 5% of the rates obtained by activated neutrophils. The composition of the NADPH oxidase of EBV-BL appears to be identical to that of phagocytic cells. In this report, methods are described for the establishment of EBV-BL, together with assays for the measurement of reactive oxidant production and detection of the constituent components of the NADPH oxidase in these cells.     

Effects of oxidative stress inhibitors, neurotoxins, and ganglioside GM1 on Na+,K+-ATPase activity in PC12 Cells and brain synaptosomes

To elucidate mechanism of ganglioside neuroprotection, it is important to study their metabolic effects, specifically of action on Na⁺,K⁺-ATPase. It has been shown that under effect of oxidative stress inductors and neurotoxins an oxidative inactivation of this enzyme takes place in PC12 cells and brain cortex synaptosomes, this inactivation being able to be prevented or decreased by ganglioside GM1. Thus, for instance, 24 h after action of 1 mM H₂O₂, activity of Na⁺,K⁺-ATPase in PC12 cells decreased more than twice. However, in the case of preincubation of the cells with ganglioside GM1 prior to the H₂O₂ action, this enzyme activity did not differ statistically significantly from control. Ganglioside GM1 also was able to increase statistically significantly the enzyme activity decreased by action on the PC12 cells of amyloid β-peptide (Aβ) causing lesion of neurons in Alzheimer’s disease and of low H₂O₂ concentrations. Experiments on brain cortex synaptosomes have established that not only antioxidants—α-tocopherol and superoxide dismutase (SOD)—but also ganglioside GM1 prevent the glutamate-produced Na⁺,K⁺-ATPase oxidative inactivation. The obtained data agree with a suggestion that the ganglioside neuroprotective effect at action on nerve cells of such toxins as Aβ, glutamate or reactive oxygen species is due to their ability to inhibit the free-radical reactions.     

Nitric oxide and antioxidant status in glucose and oxygen deprived neonatal and adult rat brain synaptosomes

Nitric oxide (NO) has been implicated in the process of cerebral ischemia/reperfusion injury. We have examined the production of NO, as reflected by nitrite (NO₂ ⁻)+nitrate (NO₃ ⁻) accumulation, from synaptosomes isolated from neonatal or adult rat brain and subjected to a period of glucose and oxygen deprivation. There was a significant increase in the amount of NO₂ ⁻+NO₃ ⁻ production from adult synaptosomes under these conditions, whereas there was no difference compared to control in the production of NO₂ ⁻+NO₃ ⁻ from the neonatal synaptosomes. The total antioxidant status of the synaptosomes at these different stages of brain development was found to be the same. These data suggest that the vulnerability of the adult brain to ischemia/reperfusion injury may be associated with the production of NO from nerve terminals. The ratios of antioxidant capacity to NO production under such conditions have been shown here to be different between the neonatal and adult nerve terminals. Thus the well documented resistance of neonatal brain to ischemia/reperfusion injury may involve the neonatal nerve terminal being under less oxidative stress than the adult.     

Protective and Antioxidative Effects of GM1 Ganglioside in PC12 Cells Exposed to Hydrogen Peroxide are Mediated by Trk Tyrosine Kinase

GM1 ganglioside was found to increase the survival of PC12 cells exposed to H₂O₂, its action was blocked by Trk tyrosine kinase inhibitor K-252a. Thus, the inhibition of H₂O₂ cytotoxic action by GM1 constituted 52.8 ± 4.3%, but in the presence of 1.0 μM K-252a it was only 11.7 ± 10.8%, i.e. the effect of GM1 became insignificant. Exposure to GM1 markedly reduced the increased accumulation of reactive oxygen species (ROS) and diminished the inactivation of Na⁺,K⁺-ATPase induced in PC12 cells by H₂O₂, but in the presence of K-252a GM1 did not change these metabolic parameters. The inhibitors of extracellular signal-regulated protein kinase, phosphatidyl inositol 3-kinase and protein kinase C decreased the effects of GM1. A combination of these protein kinase inhibitors reduced inhibition of H₂O₂ cytotoxic action by GM1 to the larger extent than each of the inhibitors and practically abolished the ability of GM1 to decrease H₂O₂-induced ROS accumulation. The protective and antioxidative effects of GM1 in PC12 cells exposed to H₂O₂ appear to be mediated by activation of Trk receptor tyrosine kinase and the protein kinases downstream from this enzyme.     

Dipeptidase activities in rat brain synaptosomes can be distinguished on the basis of inhibition by bestatin and amastatin: Identification of a kyotorphin (Tyr-Arg)-degrading enzyme

The neuropeptide kyotorphin (Tyr-Arg) was degraded by rat brain synaptosomes via a synaptic membrane-bound peptidase which was inhibited by bestatin but not by amastatin. The K_m for kyotorphin was 8×10^–6 M and the K_i for bestatin was 1×10^–7 M. The kyotorphin-degrading enzyme was distinguished from at least one other dipeptide-hydrolyzing activity in synaptosomes which was inhibited by both bestatin and amastatin. Gel permeation chromatography of detergentextracted synaptosomes resulted in the separation of the dipeptide-hydrolyzing activities. A single kyotorphin-degrading enzyme peak was observed which had a M_r=52,000. The activity peak could degrade other dipeptides including Phe-Arg, a synaptic membrane-generated metabolic of bradykinin. The kyotorphin-degrading enzyme appears to be novel and can be distinguished from other known dipeptidases on the basis of substrate specificity, subcellular localization, and inhibition profile.     

Effects of Ca2+, Mg2+, and depolarizing agents,on the32Pi-labeling and degradation of phosphatidylinositols in rat brain synaptosomes

In isolated synaptosomes from rat brain, 100 M antimycin A and 10 M oxamic acid inhibit the³²Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP₂) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95–99% respectively. 10 mM sodium fluoride inhibits the labeling by 50–60% and 10 mM A23187 inhibits the labeling by 63–70%. Phospholipase A₂ inhibits the labeling of PIP₂ and PIP by 93–94% and stimulates their degradation by 84–92%. Depolarization of synaptosomes with 75 mM K⁺ or 100 M veratrine decreases the labeling of PIP₂ and PIP by 66–74%. The decreased labeling results in large part from the Ca²⁺-dependent degradation of³²P-labeled PIP₂ and PIP as shown by pulse-chase experiments in which PIP₂ and PIP were prelabeled with³²Pi. Depolarization of synaptosomes results in the stimulation of⁴⁵Ca²⁺ uptake with the concomitant hydrolysis of PIP and PIP₂. Addition of 1 mM Ca²⁺ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K⁺ accounts for 75% of the enhanced degradation of PIP₂ and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of⁴⁵Ca²⁺ uptake. EGTA (10mM) and Mg²⁺ (5–10 mM) inhibit the degradation of PIP and PIP₂ and counteract the action of 1 mM Ca²⁺. Our data demonstrate that⁴⁵Ca²⁺, Mg²⁺, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.Abbreviations ATP adenosine triphosphate - Pi inorganic orthophosphate - PIP phosphatidylinositol-4-phosphate - PIP₂ phosphatidylinositol-4,5,-bisphosphate - IP₃ inositol-1,4,5-trisphosphate     

Choline acetyltransferase-like activity bound to neuronal plasma membranes

A form of CAT-like activity was found bound present in rat brain synaptosomal membranes which could be recovered in the Triton X-114 phase. The enzyme activity was slightly activated by NaCl, had a pH maximum around 8 and showed a temperature dependence with a Q₁₀ of 2.28. It was inhibited 100% by 10^–6 M naphthyl vinyl pyridinium but not by 10^–5 M diisopropyl phosphofluoridate. The kinetics of this bound form of CAT were similar to the soluble form of the enzyme. TheK _m was 405±58 M for choline and 62±8 M for AcCoA. Five isoelectric forms were found with pH's of 4.55, 6.05, 7.06, 7.36, and 8.00 which is in contrast to the three isoelectric forms found of the soluble enzyme in rat brain. The presence of a CAT-like activity in the plasma membrane was confirmed with experiments performed using intact synaptosomes and intact cells in culture. Acetylcholine, synthesized from radioactive AcCoA by intact rat brain synaptosomes, was recovered in the incubation medium and only in the presence of exogenous choline or when the production of choline was stimulated by oleate via the activation of phospholipase D. This was also seen in experiments with intact pheochromocytoma cell cultures (PC 12) which synthesize acetylcholine that was recoverved in the incubation medium. Acetylcholine formation in the presence of choline and AcCoA was stimulated in cells that had been grown in the presence of nerve growth factor (NGF). The localization of 1% of CAT activity in a transbilayer position in the plasma membrane, could suggest a possible role of this enzymatic form in the regulation of acetylcholine synthesis.     

Carbon turnover in peatland mesocosms exposed to different water table levels

Changes of water table position influence carbon cycling in peatlands, but effects on the sources and sinks of carbon are difficult to isolate and quantify in field investigations due to seasonal dynamics and covariance of variables. We thus investigated carbon fluxes and dissolved carbon production in peatland mesocosms from two acidic and oligotrophic peatlands under steady state conditions at two different water table positions. Exchange rates and CO₂, CH₄ and DOC production rates were simultaneously determined in the peat from diffusive-advective mass-balances of dissolved CO₂, CH₄ and DOC in the pore water. Incubation experiments were used to quantify potential CO₂, CH₄, and DOC production rates. The carbon turnover in the saturated peat was dominated by the production of DOC (10–15 mmol m^–2 d^–1) with lower rates of DIC (6.1–8.5 mmol m^–2 d^–1) and CH₄ (2.2–4.2 mmol m^–2 d^–1) production. All production rates strongly decreased with depth indicating the importance of fresh plant tissue for dissolved C release. A lower water table decreased area based rates of photosynthesis (24–42%), CH₄ production (factor 2.5–3.5) and emission, increased rates of soil respiration and microbial biomass C, and did not change DOC release. Due to the changes in process rates the C net balance of the mesocosms shifted by 36 mmol m^–2 d^–1. According to our estimates the change in C mineralization contributed most to this change. Anaerobic rates of CO₂ production rates deeper in the peat increased significantly by a factor of 2–3.5 (DOC), 2.9–3.9 (CO₂), and 3–14 (CH₄) when the water table was lowered by 30 cm. This phenomenon might have been caused by easing an inhibiting effect by the accumulation of CO₂ and CH₄ when the water table was at the moss surface.     

Metabolic effects of ganglioside GM1 on PC12 cells in oxidative stress depend on modulation of activity of tyrosine kinase Trk of receptors

There have been obtained evidences that not only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of cells of the neuronal line PC12 under action of H₂O₂. By the example of GM1 and GD1a, gangliosides have been shown to produce a protective effect on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. For the first time, GM1 at nanomolar concentrations was shown to decrease the H₂O₂-induced formation of reactive oxygen species (ROS). It was found that in the presence of inhibitor of tyrosine kinase Trk of receptors K-252a, GM1 at concentrations of 10 μM and 10 nM lost its ability to produce such metabolic effects as a decrease of ROS accumulation and of the degree of oxidative inactivation of Na⁺,K⁺-ATPase in PC12 cells, as well as ceased to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of gangliosides GM1 in PC12 cells treated with H₂O₂ on modulation of activity of activity of tyrosine kinase Trk receptors (i.e., from the same signal system) agrees with concept about the essential role of oxidant effect of GM1 in its increase of cell viability.     

Action of thiamine on different synapses

Thiamine at a concentration of 1×10^–14 to 1×10^–4 M facilitated neuromuscular transmission at the glutaminergic synapse of the crayfish adapter, manifesting as increased amplitude and quantal content of excitatory postsynaptic potentials and raised frequency of miniature excitatory postsynaptic potentials. Thiamine augmented spontaneous electrical activity and the amplitude of synaptic potentials in the longitudinal muscle of guinea pig taenia coli. It was found from studying the effects of thiamine on the membrane potential of rat brain synaptosomes that its presynaptic action is brought about by depolarization of the nerve terminal membrane. Interaction between thiamine and the nerve endings was described by a Hill coefficient of 0.22–0.30, indicating that it has several binding sites within the structure of the receptor concerned.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 621–629, September–October, 1986.     

Studies on lithium transport across the red cell membrane

Summary Binding of³H-saxitoxin to Na⁺ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1nm; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30nm. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K⁺ concentrations, or by activation of the Na⁺ channels with batrachotoxin plus a purified polypeptide toxin from the scorpionLeiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70–80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60–70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2nm. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0° of 0.1 min^–1 for membrane bound and 0.2 min^–1 for detergent-solubilized binding sites. The measured association rate constant was 6×10⁸ m ^–1 min^–1 at 0° for membrane-bound saxitoxin binding sites.     

Plankton and primary productivity of Lake Manzala,Egypt

Primary production and distribution and abundance of phyto- and zooplankton of lake Manzala were investigated from June 1985 to June 1986.Primary production varied from 4.1 to 28.7 g O₂ m^–2 d^–1 with the highest values 24.2 and 28.7 g O₂ m^–2 d^–1 recorded in the eastern and southern sectors and the lowest value 4.1 g O₂ m^–2 d^–1 recorded in the northern sector. The seasonal mean production for the lake was estimated at 13.3 g O₂ m^–2 d^–1.Mean phytoplankton abundance ranged from 32.7 10⁷ to 76.1 10⁷ cells m^–3 with a mean value of 48.10⁷ cells m^–3. Diatoms were the dominant phytoplankton group comprising 52 to 90 % by number. The greatest relative abundance (87 to 90%) was recorded in the southern sector.Mean zooplankton abundance ranged from 30.1 10³ to 44.4 10³ organisms m^–3 in the eastern sector to 5.5.10³ in the northern sector. In response of eutrophication, the species composition changed significantly over the last 20 years. Cladocerans represented less than 1% of zooplankton during 1959/60, but 75% in 1985/86. Rotifers constituted 40% in 1959/60, and only 1% in 1985/86. Cirriped larvae declined from 21% to 1%.     

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice,but not in those from gp91-phox-deficient mice

Macrophages produce superoxide (O₂^–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O₂^– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H₂DCFDA), O₂^– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H₂DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O₂^–-producing cells, but not in O₂^–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H₂O₂ dismutated from O₂^–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.     

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cellsin vitro

Recently, cadmium has been described to disturb ovarian function in rats. In this paper the direct influence of cadmium on steroid production of ovarian cellsin vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 g ml^–1 CdCl₂ in the presence or absence of 0.1–1000 ng ml^–1 follicle stimulating hormone (FSH) or luteinizing hormone (LH) for 24 or 48 h. Production of progesterone (P) and 17-estradiol (E₂) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 g ml^–1 CdCl₂ suppressed P accumulation to 65 and 10%, respectively; accumulation of E₂ (at 5 g ml^–1 CdCl₂) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 g ml^–1 CdCl₂ was added to the medium. No alteration in basal P accumulation occurred in granulosa and luteal cell cultures following incubations with 20 and 40 g ml^–1 CdCl₂, whereas basal E₂ production of granulosa cells was markedly diminished. It is concluded that CdCl₂ suppressing steroid synthesisin vitro exerts a direct influence on granulosa and luteal cell function.     

Electrical effects of potassium and bicarbonate on proximal tubule cells ofNecturus

Summary The effects of stepwise concentration changes of K⁺ and HCO ₃ ^– in the basolateral solution on the basolateral membrane potential (V _bl) of proximal tubule cells of the doubly-perfusedNecturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes inV _bl after alterations in external K⁺ concentration from 1.0 to 2.5mm (t _{K, 1.0–2.5}), 2.5 to 10, and in external HCO ₃ ^– concentration (at constant pH) from 5 to 10mm (t _{HCO3, 5–10}), 10 to 20, or 10 to 50.t _{K, 2.5–10} was 0.38±0.02 under control conditions but was sharply reduced to 0.08±0.03 (P>0.001) by 4mm Ba⁺⁺. This concentration of Ba⁺⁺ reducedV _bl by 9±1 mV (at 2.5 external K⁺). Perfusion with SITS (5×10^–4 m) for 1 hr hyperpolarizedV _bl by 10±3 mV and increasedt _{K, 2.5–10} significantly to 0.52±0.01 (P<0.001). Ba⁺⁺ application in the presence of SITS depolarizedV _bl by 22±3 mV. In control conditionst _{HCO3, 10–50} was 0.63±0.05 and was increased to 0.89±0.07 (P<0.01) by Ba⁺⁺ but was decreased to 0.14±0.02 (P<0.001) by SITS. In the absence of apical and basolateral chloride, the response ofV _bl to bicarbonate was diminished but still present (t _{HCO3, 10–20} was 0.35±0.03). Intracellular pH, measured with liquid ion-exchange microelectrodes, increased from 7.42±0.19 to 7.57±0.17 (P<0.02) when basolateral bicarbonate was increased from 10 to 20mm at constant pH. These data show that the effects of bicarbonate onV _bl are largely independent of effects on the K⁺ conductance and that there is a significant current-carrying bicarbonate pathway in the basolateral membrane. Hence, both K⁺ and HCO ₃ ^– gradients are important in the generation ofV _bl, and their relative effects vary reciprocally.     

Relative stability of α-melanotropin and related analogues to rat brain homogenates

α-Melanotropin (α-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle⁴, D-Phe⁷]-α-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–10-NH₂, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–11-NH₂ is totally resistant to inactivation by rat brain homogenate. Both [Nle⁴, D-Phe⁷]-fragments are resistant to degradation by rat serum, but [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH_4–10-NH₂ and Ac-[Nle⁴]-α-MSH_4–11-NH₂ are rapidly inactivated under both conditions. The cyclic melanotropin, [ ]-α-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[ ]-α-MSH_4–10-NH₂ analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[ ]-α-MSH_4–10-NH₂ is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Characteristics of an acid active thiamine diphosphatase from beef brain

That thiamine has a role in nerve conduction as well as synaptic transmission is suggested by the following observations. (1) Thiamine phosphate esters are hydrolyzed and released from nerve membranes during nerve conduction. (2) Ultraviolet radiation of single nerve fibers at the wavelength specific for thiamine destroys the ability of that nerve to conduct an impulse. (3) Thiamine diphosphatase (TDPase) is present on synaptosomes. Previous articles have characterized an alkaline active TDPase in brain; this report characterizes a pH 5 active TDPase and compares its properties to the pH 9 enzyme. Both enzymes require a divalent cation for optimal activity. The pH 5 enzyme is more sensitive to ATP. Myelin fractions of brain have the highest specific activity for the acid TDPase, and the nerve ending particles the highest total activity. No PO₄ ^3– inhibition was observed. Kinetic constants of this enzyme activity are reported.