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Interactions of bilirubin with isolated presynaptic nerve terminals: Functional effects on the uptake and release of neurotransmitters

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Summary

  1. 1.

    The functional effects of bilirubin:albumin solutions (10:1, mol/mol) on several synaptosomal functions were investigated using rat cortical, striatal, and hippocampal synaptosomes prepared by iso-osmotic Percoll/sucrose gradient centrifugation.

  2. 2.

    Bilirubin (10–80µM) depolarized synaptosomes in a tetrodotoxin-insensitive manner as assessed by the equilibrium distribution of tetra-[3H]phenylphosphonium. Depolarization induced by bilirubin was of a lesser magnitude than that caused by KCl or veratridine. Steady-state pH gradients across the synaptosomal membrane were determined using the transmembrane distribution of [14C]methylamine. Bilirubin (20–40µM) did not modify the intracellular pH in physiological buffers. The pigment effected a 0.14 ΔpH change when the synaptosomes were suspended in a Ca2+ and Na+ free choline medium containing ouabain.

  3. 3.

    Bilirubin (20–80µM) had no effect of its own on [7,8-3H] dopamine release from striatal synaptosomes. In contrast, it inhibited the initial rate of synaptosomal uptake of the catecholamine and its intrasynaptosomal content at 10 min. The pigment (20 and 40µM) reduced the 35 mM KCl-induced release of endogenous acetylcholine from hippocampal synaptosomes by 20 and 36%, respectively.

  4. 4.

    The association of bilirubin with synaptic plasma membrane vesicles was characterized by a chloroform:methanol 2:1 (v/v) extraction method. At total concentrations of 10 to 80µM bilirubin, the molar percentage of the pigment in synaptic plasma membrane phospholipids was 1–4%.

  5. 5.

    It is proposed that the two main functional consequences of the bilirubin-nerve ending interaction are an impairment of specific membranebound neurotransmitter uptake mechanisms and a reduction of the response to depolarizing stimuli. This may be the basis for rapid alterations in synaptic transmission documented in early reversible bilirubin encephalopathy.

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References

  • Aiuchi, T., Matsunaga, M., Nakaya, K., and Nakamura, Y. (1989). Calculation of membrane potential in synaptosomes with use of a lipophilic cation (tetraphenylphosphonium).Chem. Pharm. Bull. (Tokyo)373333–3337.

    Google Scholar 

  • Amit, Y., Chan, G., Fedunec, S., Poznansky, M. J., and Schiff, D. (1989). Bilirubin toxicity in a neuroblastoma cell line N-115. I. Effects on Na+K+ ATPase, [3H]-thymidine uptake, L-[35S]methionine incorporation, and mitochondrial function.Pediat. Res. 25364–368.

    Google Scholar 

  • Amit, Y., Fedunec, S., Thomas, P. D., Poznansky, M. J., and Schiff, D. (1990). Bilirubin-neural cell interaction: Characterization of initial cell surface binding leading to toxicity in the neuroblastoma cell line N-115.Biochim. Biophys. Acta 105536–42.

    Google Scholar 

  • Bahler, M., Benfenati, F., Valtorta, F., and Greengard, P. (1990). The synapsins and the regulation of synaptic function.BioEssays 12259–263.

    Google Scholar 

  • Blaustein, M. P., and Goldring, J. M. (1975). Membrane potentials in pinched-off presynaptic nerve terminals monitored with a fluorescent probe: Evidence that synaptosomes have potassium diffusion potentials.J. Physiol. (Lond.)247589–615.

    Google Scholar 

  • Bratlid, D. (1990). How bilirubin gets into the brain.Clin. Perinatol. 17449–465.

    Google Scholar 

  • Breckenridge, W. C., Gombos, G., and Morgan, I. G. (1972). The lipid composition of adult rat brain synaptosomal plasma membranes.Biochim. Biophys. Acta 266695–707.

    Google Scholar 

  • Brodersen, R., and Stern, L. (1990). Deposition of bilirubin acid in the central nervous system—a hypothesis for the development of kernicterus.Acta Paediat. Scand. 7912–19.

    Google Scholar 

  • Cathcart, R., Schwiers, E., and Ames, B. N. (1983). Detection of picomole levels of hydrogen peroxides using a fluorescent dichlorofluorescein assay.Anal. Biochem. 134111–116.

    Google Scholar 

  • Deutsch, C., and Rafalowska, U. (1979). Transmembrane electrical potential measurements in rat brain synaptosomes.FEBS Lett. 108274–278.

    Google Scholar 

  • Deutsch, C., Dron, C., Rafalowska, U., and Silver, I. A. (1981). Synaptosomes from rat brain: morphology, compartmentation, and transmembrane pH and electrical gradients.J. Neurochem. 362063–2072.

    Google Scholar 

  • Drapeau, P., and Nachsen, D. (1988). Effects of lowering extracellular and cytosolic pH on calcium fluxes, cytosolic calcium levels, and transmitter release in presynaptic nerve terminals isolated from rat brain.J. Gen. Physiol. 91305–315.

    Google Scholar 

  • Eriksen, E., Danielsen, H., and Brodersen, R. (1981). Binding of Bilirubin dianion, protonization, and aggregation of bilirubin acid.J. Biol. Chem. 2564269–4274.

    Google Scholar 

  • Glowinski, J., and Iversen, L. L. (1966). Regional studies of catechloamines in the rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine, and [3H]dopa in various regions of the brain.J. Neurochem. 13655–669.

    Google Scholar 

  • Hansen, T. W., Bratlid, D., and Walaas, S. I. (1988a). Bilirubin decreases phosphorylation of synapsin I, a synaptic vesicle-associated neuronal phosphoprotein, in intact synaptosomes from rat cerebral cortex.Pediat. Res. 23219–223.

    Google Scholar 

  • Hansen, T. W., Paulsen, O., Gjerstad, L., and Bratlid, D. (1988b). Short-term exposure to bilirubin reduces synaptic activation in rat transverse hippocampal slices.Pediat. Res. 23453–456.

    Google Scholar 

  • Hansson, E., Jacobsen, I., Venema, R., and Selstrom, A. (1980). Measurement of the membrane potential of isolated nerve terminals by the lipophilic cation [3H]triphenylmethylphosphonium bromide.J. Neurochem. 42569–573.

    Google Scholar 

  • Karp, W. B. (1979). Biochemical alterations in neonatal hyperbilirubinemia and bilirubin encephalopathy: A review.Pediatrics 64361–368.

    Google Scholar 

  • Kongsamut, S., and Nachsen, D. A. (1988). Mesurement of the cytosolic sodium ion concentration in rat brain synaptosomes by a fluorescence method.Biochim. Biophys. Acta 940241–246.

    Google Scholar 

  • Leonard, M., Noy, N., and Zakim, D. (1989). The interactions of bilirubin with model and biological membranes.J. Biol. Chem. 2645648–5652.

    Google Scholar 

  • Lichtshtein, D., Kaback, H. R., and Blume, A. J. (1979). Use of a lipophilic cation for determination of membrane potential in neuroblastomaglioma hybrid cell suspensions.Proc. Natl. Acad. Sci. USA 76650–654.

    Google Scholar 

  • Lowry, O. H., and Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193265–275.

    Google Scholar 

  • MacDonald, R. C. (1989). A fluorometric assay for acetylcholine with picomole sensitivity.J. Neurosci. Methods 2973–76.

    Google Scholar 

  • Mayor, F., Jr., Pages, M., Diez-Guerra, J., Valdivieso, F., and Mayor, F. (1985). Effect of postnatal anoxia on bilirubin levels in rat brain.Pediat. Res. 19231–236.

    Google Scholar 

  • Mayor, F., Jr., Diez-Guerra, J., Valdivieso, F., and Mayor, F. (1986). Effect of bilirubin on the membrane potential of rat brain synaptosomes.J. Neurochem. 47363–369.

    Google Scholar 

  • McClare, C. W. F. (1971). An accurate and convenient organic phosphorus assay.Anal. Biochem. 39527–530.

    Google Scholar 

  • Mustafa, M. G., and King, T. E. (1970). Binding of bilirubin with lipid. A possible mechanism of its toxic reactions in mitochondria.J. Biol. Chem. 2451084–1089.

    Google Scholar 

  • Nachsen, D. A., and Drapeau, P. (1988). The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain.J. Gen. Physiol. 91289–304.

    Google Scholar 

  • Nagaoka, S., and Cowger, M. L. (1978). Iteraction of bilirubin with lipids studied by fluorescence quenching method.J. Biol. Chem. 2532005–2011.

    Google Scholar 

  • Nagy, A., and Delgado-Escueta, A. V. (1984). Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll).J. Neurochem. 431114–1123.

    Google Scholar 

  • Ochoa, E. L. M., Chattopadhyay, A., and McNamee, M. G. (1989). Desensitization of the nicotinic acetylcholine receptor: molecular mechanisms and effect of modulators.Cell. Mol. Neurobiol. 9141–178.

    Google Scholar 

  • Ochoa, E. L. M., Li, L., and McNamee, M. G. (1990). Desensitization of central cholinergic mechanisms and neuroadaptation to nicotine.Mol. Neurobiol. 4251–287.

    Google Scholar 

  • Ochoa, E. L. M., Nguyen, T., Wennberg, R. P., and Takashima, T. (1991). Effects of bilirubin on transmembrane electrical and proton gradients of rat cortical synaptosomes and on reconstituted nicotinic acetylcholine receptor function.Soc. Neurosci, Abstr. 171460.

    Google Scholar 

  • Ramos, S., Grollman, E. F., Lazo, P. S., Dyer, S. A., Habig, W. H., Hardegree M. C., Kaback, H. R., and Kohn, L. D. (1979). Effect of tetanus toxin on the accumulation of the permeant lipophilic cation tetraphenylphosphonium by guinea pig brain synaptosomes.Proc. Natl. Acad. Sci. USA 764783–4787.

    Google Scholar 

  • Reith, M. E., Jacobson, A. E., Rice, K. C., Benuck, M., and Zimanyi, I. (1991). Effect of metaphit on dopaminergic neurotransmission in rat striatal slices: Involvement of the dopamine transporter and voltage-dependent sodium channel.J. Pharmacol Exp. Ther. 2591188–1196.

    Google Scholar 

  • Sauvaigo, S., Vigne, P., Frelin, C., and Lazdunski, M. (1984). Identification of an amiloride-sensitive Na+/H+ exchange system in brain synaptosomes.Brain Res. 301371–374.

    Google Scholar 

  • Schmalzing, G. (1987). Proton permeability of the plasma membrane of rat cortical synaptosomes.Eur. J. Biochem. 16827–35.

    Google Scholar 

  • Schmalzing, G., Kutschera, P., and Schlosser, T. (1986). Li+ as substrate of the synaptosomal Na+/H+ antiporter.J. Biol. Chem. 2612759–2767.

    Google Scholar 

  • Scott, I. D., and Nicholls, D. G. (1980). Energy transduction in intact synaptosomes. Influence of plasma membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.Biochem. J. 18621–33.

    Google Scholar 

  • Tkachenko, A. V. (1988). Effect of bilirubin and serum albumin on the Na,K-ATPase activity in the synaptosomal membrane.Tsitologiia 30849–854.

    Google Scholar 

  • Tkachenko, A. V. (1989). Functional changes in the synaptic membrane during in vitro interaction with bilirubin and serum albumin.Vopr. Med. Khim. 3539–44.

    Google Scholar 

  • Vázquez, J., Garcia, C. M., Valdivieso, F., Mayor, F., and Mayor, F., Jr. (1988). Interaction of bilirubin with the synaptosomal plasma membrane.J. Biol. Chem. 2631255–65.

    Google Scholar 

  • Vázquez, J., Ortega, G., Valdivieso, F., and Mayor, F. J. (1989). Interaction of bilirubin with gangliosides.J. Biochem. (Tokyo)106139–142.

    Google Scholar 

  • Walaas, S. I., and Greengard, P. (1991). Protein phosphorylation and neuronal function.Pharmacol. Rev. 43299–349.

    Google Scholar 

  • Wennberg, R. P. (1988). The importance of free bilirubin acid salt in bilirubin uptake by erythrocytes and mitochondria.Pediat. Res. 23443–447.

    Google Scholar 

  • Wennberg, R. P., Ahlfors, C. E., Bickers, R., McMurtry, C. A., and Shetter, J. L. (1982). Abnormal auditory brainstem response in a newborn infant with hyperbilirubinemia: Improvement with exchange transfusionJ. Pediat. 100624–626.

    Google Scholar 

  • Yang, B. J., Morris, M. D., Xie, M. Q., and Lightner, D. A. (1991). Resonance Raman spectroscopy of bilirubins: Band assignments and application to bilirubin/lipid complexation.Biochemistry 30688–694.

    Google Scholar 

  • Zakim, D., and Wong, P. T. (1990). A high-pressure, infrared spectroscopic study of the solvation of bilirubin in lipid bilayers.Biochemistry 292003–2007.

    Google Scholar 

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Authors and Affiliations

  1. Department of Pediatrics, School of Medicine, University of California at Davis, 95616, Davis, California, USA

    Enrique L. M. Ochoa, Richard P. Wennberg, Yan An & Tarun Tandon

  2. Department of Biochemistry and Biophysics, University of California at Davis, 95616, Davis, California, USA

    Enrique L. M. Ochoa, Yan An, Tom Takashima, Tom Nguyen & Alan Chui

  3. Department of Pediatrics, Division of Neonatology, TB-193, UCD Campus, University of California at Davis, 95616, Davis, California, USA

    Enrique L. M. Ochoa

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  1. Enrique L. M. Ochoa
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Ochoa, E.L.M., Wennberg, R.P., An, Y. et al. Interactions of bilirubin with isolated presynaptic nerve terminals: Functional effects on the uptake and release of neurotransmitters. Cell Mol Neurobiol 13, 69–86 (1993). https://doi.org/10.1007/BF00712990

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