Continuous enzyme-linked fluorometric detection of L-(+)-lactate released from rat brain vesicles under anoxic...

A method is described for the on-line detection of L-(+)-lactate released from brain vesicles under physiological conditions. The principle of L-lactate detection is based on the reversible oxidation of L-lactate catalysed by L-lactate dehydrogenase (LDH, EC 1.1.1.27) employing 3-acetylpyridine-adenine-dinucleotide (APAD) as analogue of NAD according to the reaction: L-lactate + APAD reversible pyruvate + APADH. In practical terms, L-lactate synthesis of vesicles incubated in the presence of LDH and APAD was continuously followed by the fluorescence (490 nm) of APADH excited at 410 nm. Addition of a L-lactate standard (10 mumol/l) enhanced APADH fluorescence with a half-life of 6.0 +/- 0.6 s allowing us to uncover a short-term alteration of L-lactate synthesis. This method was applied to evaluate a prospective change of L-lactate generation caused by the anoxia-induced increase in intravesicular Na+ and Ca2+ concentration ([Na+]i, [Ca2+]i), both fluorometrically determined by SBFI and Fura, respectively. Upon anoxia, [Na+]i and [Ca2+]i increased continuously up to 40 mmol/l Na+ and 900 nmol/l Ca2+ within 400 s. Concurrently, intravesicular NADH ([NADH]i) and basal L-lactate synthesis were enhanced within a few seconds, the latter from 4.2 +/- 1.5 to 15.8 +/- 1.5 nmol L-lactate/min per mg protein. Incubation of vesicles in the presence of 10 mumol/l tetrodotoxin (TTX) suppressed the increase in [Na+]i and [Ca2+]i but failed to influence L-lactate synthesis. The data indicate a continuous Na+ influx via voltage-dependent Na+ channels accompanied by an increase in [Ca2+]i during anoxia which did not affect anaerobic L-lactate synthesis. The method of fluorometric L-lactate determination was confirmed to be suitable for the detection of L-lactate released under physiological conditions from brain vesicles and seems to be applicable to various cell models.