Application of a novel radioimmunoassay for serotonin in the detection of aromatic l-amino acid decarboxylase activity in isolated renal cortical cells

Abstract

The decarboxylation of amino acids is an cssential step in the biosynthesis of biogenic amines; the enzyme aromatic Lamino acid decarboxylase (AAAD; EC 4.1.1.28) catalyses the decarboxylation of both ~-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan (5-HTP) to form dopamine or serotonin (5-HT), respectively. The AAAD enzyme is present in many tissues (Rahman et ul., 1 9 8 1 ), but is particularly active in the kidney, where the products of decarboxylation (i.e. dopamine and 5-HT) are thought to affect renal haemodynamics directly. Dopamine causes marked natriuresis, diuresis, and dilatation of the renal vasculature, whereas 5-HT is thought to exert opposite effects (Stier et al., 1984). Several previous studies have measured AAAD activity by incubating tissue homogenates with one of the enzyme substrates, followed either by direct measurement of the mass of product generated in the incubate, using high-pressure liquid chromatography coupled with electrochemical detection (Stier et al., 1987) or by measuring the amount of C 0 2 produced during the decarboxylation reaction (Clark et d., 1954). Both techniques are time consuming, and therefore restrict the throughput of large numbers of samples. We have used a radioimmunoassay ( M A ) for 5-HT developed in our own laboratories (Gow et al., 1987) to measure the production in vitro of 5-HT from 5-HTP by isolated rat renal cortical cells (RCC). Female Wistar rats (200-250 g) were killed by cervical dislocation and the kidneys removed immediately into icecold physiological saline. The kidneys were trimmed of fat, the capsules removed, and the cortex dissected from the medulla. Intact RCC were prepared using the collagenase digestion procedure described by Drury et ul. (1986), with modifications by Noble et al. (1988). The cells were finally resuspended in Medium 199 containing bovine serum albumin ( 2 g/l). For each experiment, aliquots (800 pl ) of a freshly prepared RCC suspension containing 0.5 x 1 O6 cells/ml were dispensed in duplicate into glass tubes in an ice/water slurry (4C), followed by Medium 199 (100 pl) containing both pargyline (final concentration 1 W 6 mol/l), and varying doses of 5-HTP ( O l O s mol/l, final concentration). A further aliquot of Medium 199 (100 p l ) or, for the inhibitor experiments, Medium 199 containing the AAAD inhibitor carbidopa (100 pl; lo-' mol/l, final concentration) was added, and the tubes incubated in a shaking water-bath (120 cycles/min) at 37°C for 30 min. The reaction was stopped by plunging the incubation tubes into an ice/water slurry, followed by centrifugation at 1720 g for 20 min at 4°C. The supernatants were then collected, and frozen at 20°C until analysis by RIA. Our results (Fig. 1) show a dose-dependent production of 5-HT by RCC in response to the increasing concentration of substrate (5-HTP). This response was significantly diminished ( P < 0.05, 2-way analysis of variance) by the inclusion of carbidopa ( lo- mol/l) in the incubates and is in agreement with the observations of Stier & ltskovitz (1985).