Glycine receptors in cultured chick sympathetic neurons are excitatory and trigger neurotransmitter release.
1. Total RNA isolated from embryonic chick paravertebral sympathetic ganglia was used in a reverse transcription-polymerase chain reaction (RT-PCR) assay with a pair of degenerate oligonucleotide primers deduced from conserved regions of mammalian glycine receptor alpha-subunits. Three classes of cDNA were identified which encode portions of the chicken homologues of the mammalian glycine receptor alpha 1, alpha 2 and alpha 3 subunits. 2. The presence of functional glycine receptors was investigated in the whole-cell configuration of the patch-clamp technique in neurons dissociated from the ganglia and kept in culture for 7-8 days. In cells voltage clamped to -70 mV, glycine consistently induced inward currents in a concentration-dependent manner and elicited half-maximal peak current amplitudes at 43 microM. 3. The steady-state current-voltage relation for glycine-induced currents was linear between +80 and -60 mV, but showed outward rectification at more hyperpolarized potentials. Reversal potentials of these currents shifted with changes in intracellular chloride concentrations and matched the calculated Nernst potentials for chloride. 4. beta-Alanine and taurine were significantly less potent than glycine in triggering inward currents, with half-maximal responses at 79 and 86 microM, respectively. At maximally active concentrations, beta-alanine-evoked currents were identical in amplitude to those induced by glycine. Taurine-evoked currents, in contrast, never reached the same amplitude as glycine-induced currents. 5. The classical glycine receptor antagonist strychnine reversibly reduced glycine-induced currents, with half-maximal inhibition occurring at 62 nM. Two more recently characterized glycine receptor antagonists, isonipecotic acid (half-maximal inhibition at 2 mM) and 7-trifluoromethyl-4-hydroxyquinoline-3-carboxylic acid (half-maximal inhibition at 67 microM), also blocked glycine-evoked currents in a reversible manner. The chloride channel blocker picrotoxin reduced glycine-evoked currents, with half-maximal effects at 348 microM. Inhibition by the glycine receptor channel blocker cyanotriphenylborate was half-maximal at 4 microM. 6. Apart from evoking inward currents, glycine occasionally triggered short (< 100 ms) spike-like currents which were abolished by hexamethonium and thus reflected synaptic release of endogenous acetylcholine. In addition, glycine caused Ca(2+)-dependent and tetrodotoxin-sensitive tritium overflow from neurons previously labelled with [3H]noradrenaline. This stimulatory action of glycine was reduced in the presence of strychnine and after treatment with the chloride uptake inhibitor furosemide (frusemide). 7. In 65% of neurons loaded with the Ca2+ indicator fura-2 acetoxymethyl ester, glycine increased the ratio of the fluorescence signal obtained with excitation wavelengths of 340 and 380 nm, respectively, which indicates a rise in intracellular Ca2+ concentration. 8. The results show that sympathetic neurons contain transcripts for different glycine receptor alpha-subunits and carry functional heteromeric glycine receptors which depolarize the majority of neurons to trigger transmitter release.
Boehm
S
S
Max-Planck-Institut für Hirnforschung, Abteilung Neurochemie, Frankfurt/Main, Germany.
Harvey
R J
RJ
von Holst
A
A
Rohrer
H
H
Betz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D004558:Electric Stimulation; D004594:Electrophysiology; D018684:Glycine Agents / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D018009:Receptors, Glycine / Q000187:drug effects / Q000378:metabolism*; D013331:Strychnine / Q000494:pharmacology; D013564:Sympathetic Nervous System / Q000187:drug effects / Q000378:metabolism*
D018684:Glycine Agents; D018377:Neurotransmitter Agents; D018009:Receptors, Glycine; D013331:Strychnine; D002118:Calcium; D009638:Norepinephrine