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

Abstract

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 ⇌ 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 μmol/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 Ca 2+ concentration ([Na +] i, [Ca 2+] i), both fluorometrically determined by SBFI and Fura, respectively. Upon anoxia, [Na +] i and [Ca 2+] i increased continuously up to 40 mmol/l Na + and 900 nmol/l Ca 2+ 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 μmol/l tetrodotoxin (TTX) suppressed the increase in [Na +] i and [Ca 2+] 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 [Ca 2+] 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.