An improved method for the determination of pyridoxal-5-phosphate using tyrosine decarboxylase is described.Pyridoxal-5-phosphate (PLP) is the coenzyme form of vitamin B6 for a multiplicity of biosynthetic and catabolic enzyme systems. The methods generally used for the determination of PLP in biological samples are either enzymatic or chemical procedures. The method described here is basically the same as other enzymatic procedures (Bhagavan et al., 1976;Chabner and Livingston, 1970;Reinken, 1972), using radioactive tyrosine and the apoenzyme, tyrosine decarboxylase from Streptococcusfaecalis. However, instead of collecting ]4CO2, the other product of the enzymatic reaction, [3H]tyramine, was determined after separation from the substrate, [3H]tyrosine, by a mini DEAE-cellulose column.The assay mixture for PLP measurement contained 0.1 mol/l sodium acetate buffer, pH 5.5, the enzyme, tyrosine decarboxylase (approximately 20mU), PLP (10-300pg) and [aH]tyrosine (0.5~tCi/0.25pmol) in a total volume of 0.3 ml. The reaction mixture was preincubated in the absence of tyrosine for 20 min to allow the formation of the holo enzyme. The reaction was then initiated by addition of tyrosine, incubated for 15 min at 37°C, and terminated by heating the mixture for 3 min at 95 °C. After cooling the mixture in an ice bath, 100 lal of the supernatant was loaded on to a mini DEAEcellulose column (0.9 x 1.0cm) prepared with a 2.5 ml disposable syringe. The preparation of the OH form of

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Key words: pH vagus nerve ion-sensitive micro-electrode ouabain excitability rat Double-barrelled pH-sensitive micro-electrodes were used to record changes of extracellular pH during repetitive stimulation of isolated rat vagus nerves. It was found that a small initial alkaline shil) was followed by a prolonged acidification. The acidification was correlated in time with the poststimulus undershoot o[" the extracellular K + activity and with the recovery phase of the nerve conduction velocity. In the presence of ouabain, the acid component of the pH change was completely abolished (indicating a metabolic origin), whereas the alkaline component remained unaltered. These pH changes were too small to make a significant contribution to the activity-related changes in conduction velocity of the vagal ('-fibres.Activity in central and peripheral nerve fibres is accompanied by alterations of their excitability. The possible contribution of changes in extracellular pH (pHi.) to modifications of axonal excitability has, however, remained a matter of speculation. A recently designed neutral carrier-based hydrogen ion-selective micro-electrode [1] has now enabled us to obtain information about activity-related pH changes in rat ventral roots and vagus nerves. Whereas activity of the myelinated nerve fibres in the spinal roots was not accompanied by a detectable (i.e. bigger than 0.01 pH units) rise or decrease of pH~ [4], unmyelinated nerve fibres in rat vagus nerves did show such alterations. The data from the latter preparation are described in the present paper.Cervical vagus nerves were isolated from urethane-or thiogenal-anesthetized rats and the connective tissue sheath was removed. The experiments were performed in a small temperature-controlled perspex bath (25-30 C) through which a solution of the following composition was pumped (in mM): NaCI 118, KC1 3, CaCI: 1.5, NaHCO~ 25, NaH2PO4 1.2, MgCl21, glucose 10. The free nerve ends were drawn into a pair of glass suction electrodes which allowed stimulation and recording of compound potentials. Stimulus strength was adjusted above the maximal amplitude of the C-fibre component. Double-barrelled pH-(Fluka 82500) and K +-sensitive (Corn-*Author for correspondence.tl3114-3940'86~$ 03.50 ~)

Double-barrelled pH-sensitive micro-electrodes were used to measure changes of intracellular and extracellular pH in and around motoneurons of the isolated frog spinal cord during application of excitatory amino acids. It was found that N-metbyl-D-aspartate, quisqualate and kainate produced a concentrationdependent intracellular acidification. Extracellularly, triphasic pH changes (acid-alkaline-acid going pH transients) were observed during the action of these amino acids. The possible significance of such pH changes for the physiological and pathophysiological effects of excitatory amino acids are discussed.Amongst the acidic amino acid receptors in the nervous system the N-methyl-Daspartate (NMDA)-preferring subtype has obtained particular interest due to its possible involvement in phenomena such as long-term potentiation, epilepsy and neurodegeneration (see ref. 7 for a review). It has been described that the NMDA receptor-activated channel is highly permeable for Ca 2+, [5, 10,12] in contrast to channels activated by quisqualate and kainate. Consequently, the rise in intracellular Ca 2+ may serve as a mediator for secondary changes of neuronal excitability. Another possible factor, however, by which NMDA receptors could alter neuronal excitability may be a change in extra-and/or intracellular pH. To our knowledge, this possibility has not yet been explored. Therefore, we have investigated how acidic amino acids influence the extracellular (pile) and intracellular pH (pHi) around and in motoneurons of the isolated frog spinal cord. Glutamate-induced changes of other intracellular ion activities in frog motoneurons have been already described [3].Experiments were done on frog spinal cords [8,9]. After decapitation, a ventral laminectomy was performed in cooled Ringer solution. The spinal cord, including dorsal and ventral roots of the lumbar segments, was removed and placed in a recording chamber (volume 1.5 ml), which was continuously superfused with Ringer solution by means of a roller pump (2.5 ml/min). The Ringer solution contained (mM): NaCI 98.0, KC1 3.6, CaCI2 2.0, NaHCO3 12.0, glucose 10. Note the absence of Mg 2 t in the Ringer solution. Addition of 1 mM Mg 2+, in accordance with Ault

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