Bovine adrenal glands were obtained from local slaughterhouses. They were carried to the laboratory chilled in an ice bath. The medullae were digested by retrograde perfusion through the central vein with collagenase B (Roche Molecular Biochemicals / Boehringer Mannheim Biochemicals, Mannheim, Germany), and the medullary suspension was purified by centrifugation through a meglumine diatrizoate - sodium diatrizoate gradient (made in the laboratory, or available commercially as Urografin® from Schering AG, Berlin, Germany). Purification was continued by differential plating in tissue culture flasks for two to three hours. The cells were plated on appropriate dishes or plates (Wilson and Viveros, 1981; Waymire et al., 1983; Wilson, 1987; Tuominen et al., 1991), grown in standard medium (Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 with 10% foetal calf serum and antibiotics), and used within a week of preparing the culture. The cultures yielded 10 - 120 x 106 cells per gland. Despite differences in cell yield, cultures were consistently of 90% purity as estimated by neutral red staining.
The cells were labelled with [3H]oleic acid or [3H]myristic acid (both from Amersham, Buckinghamshire, UK) for 24 h. They were then washed with isotonic saline and gently suspended in an appropriate buffer (pH 7.4 at 37°C): based on bicarbonate (5% CO2 atmosphere) or on N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES, room air). The assay was based on a PLD-catalysed characteristic reaction, where primary alcohol replaces water in accepting the phosphatidyl moiety. The phosphatidylalcohol thus formed is more stable than phosphatidic acid and can be used as a measure of PLD activity (see Morris et al., 1997a). Hence, the cells were exposed to stimulants in the presence of 50 mM n-butanol (I) or 220 mM ethanol (II). In 1-h exposures, the alcohol was present for the last 15 min.
The lipids were extracted with chloroform : methanol : water (10 : 10 : 9, by volume), acidified with HCl (Bligh and Dyer, 1959). The chloroform phase was collected, the solvent was evaporated, and the lipid residue was redissolved in chloroform : methanol (9 : 1). The phospholipids were then separated by thin layer chromatography (LK6D plates from Whatman, Clifton, NJ, USA). The solvent used for phosphatidylbutanol separation was the upper phase of ethylacetate : isooctane : acetic acid : water (13 : 2 : 3 : 10) (Liscovitch and Amsterdam, 1989). When phosphatidylethanol was formed, oxalate-coated thin layer chromatography plates were used with a chloroform : methanol : acetic acid : water (75 : 45 : 3 : 1) mixture as the solvent (Horwitz, 1991; Caumont et al., 1998). Phosphatidylalcohol markers were run along with the samples. The phospholipids on chromatography plates were stained with iodine.
Phosphatidylethanol and phosphatidylbutanol standards were prepared from 1-palmitoyl-2-oleyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA) by Streptomyces sp. PLD (Sigma, St. Louis, MO, USA), as previously described (Eibl and Kovatchev, 1981; Kobayashi and Kanfer, 1987; Comfurius et al., 1990). A two-phase system consisting of 50 ml l-1 n-butanol or ethanol, 450 ml l-1 chloroform, and 500 ml l-1 185 mM Tris buffer (pH 7.4) with 185 mM CaCl2, was used. The mixture was shaken for 2 - 4 h at 37°C. The lipids were extracted as above (Bligh and Dyer, 1959).
The cultured cells were washed and homogenised by sonication in the presence of protease inhibitors phenylmethylsulfonyl fluoride 1 mM and leupeptin 250 g ml-1. The protein extracts were prepared by high-acceleration centrifugation (100 000 x g, 45 - 60 min), first in the absence and then in the presence of 0.1% Triton X-100 to extract the soluble and membrane proteins, respectively. The extracts were denatured, and the protein samples were separated in 8% polyacrylamide gels (Laemmli, 1970) and transferred to nitrocellulose membranes (Schleicher & Shüll, Dassel, Germany) by semi-dry electroblotting (SemiPhor™, Hoefer Pharmacia Biotech, San Fransisco, CA, USA). The membranes were blocked in 5% fat-free milk, exposed to antibodies, and the immunoreactive proteins were detected by enhanced chemiluminescence reaction (Durrant, 1990; Crisp and Dunn, 1994).
Rabbit anti-rat PKC antisera were a generous gift from Dr. William C. Wetsel (NIEHS, NC, USA). They were raised against carboxyterminal peptides of PKC isoenzymes as follows: SYVNPQFVHPILQSAV (a), SYTNPEFVINV (b1), SFVNSEFLKPEVKS (b2), DARSPTSPVPVPVM (g), SFVNPKYEQFLE (d), KGFSYFGEDLMP (e), EYINPLLLSAEESV (z), and their preparation and properties have previously been described (Wetsel et al., 1992). Anti-PLD antibody, against sequence CIIGSANINERS, was a product of Upstate Biotechnology, Lake Placid, NY, USA. Goat anti-rabbit secondary antibody was purchased from Bio-Rad Laboratories, CA, USA.
The protein concentrations of the extracts were determined according to Bradford (1976). The dye reagent was either commercial (III, IV; Bio-Rad, Richmond, CA, USA), or it was prepared from Serva Blue G (I, VI; Serva, Heidelberg, Germany). The protein standards were prepared from bovine serum albumin concentrate (Pierce, Rockford, IL, USA).
The experiments were run largely as previously described (McKay and Schneider, 1984), in saline buffered with HEPES and NaHCO3 (pH 7.4 at 5% CO2). Ascorbic acid 1 mg ml-1 was added to the medium to avoid oxidation of the catecholamines. To get a lower non-specific release, 1 mg ml-1 bovine serum albumin was added in some experiments (II, IV). The cell catecholamine pools were labelled with 500 nM [3H]noradrenaline (Amersham, 22 kBq ml-1) for 10 min (II, IV) or 60 min (III). Then, the cells were washed and exposed to stimulants as indicated. The treatments were terminated by pipetting the medium into scintillation vials or plates. The cells were disrupted in 0.1% Triton X-100 before transferring them to scintillation vials or plates. Results are expressed as percentages of total cellular [3H]noradrenaline contents released.
The cells were washed and gently resuspended in experiment buffer (NaCl 118 mM, KCl 5 mM, MgSO4 1 mM, CaCl2 1 mM, HEPES 20 mM, glucose 10 mM, pH 7.4). The cells were labelled with 5 M Fura 2-AM (Molecular Probes, Eugene, OR, USA) for the first 10 min, and then with 500 nM Fura 2-AM for an additional hour (McMillian et al., 1992). Then, the cells were washed, incubated for at least 10 min and washed again. All the incubations were performed in the dark to avoid photobleaching. For experiments in a Ca++-free environment, CaCl2 was omitted from the experiment buffer and 100 M ethylene glycol-bis(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) was added. Measurements were performed at room temperature in a quartz cuvette (2 x 106 cells/ml), with a Hitachi F2000 fluorometer essentially as previously described (Törnquist et al., 1999). The excitation wavelengths were 340 nm and 380 nm, and the emission wavelength was 510 nm. [Ca++]i was calculated as previously described (Grynkiewicz et al., 1985), using a software designed for the fluorometer. The Kd value for Fura 2 was 224 nM.
The cells were exposed to the test compounds for the desired time. In the 24-hour experiments, 0.5 mg ml-1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma, St. Louis, MO, USA) was added three hours before stopping the treatment. In short experiments, MTT was present throughout the incubation. For measuring toxicity, media were aspirated and replaced with dimethyl sulfoxide. After shaking for 5 min, the absorbances were read at 540 nm to estimate the MTT reduction. The extent of reduction was considered as a measure of mitochondrial function and cell survival (Supino, 1990).
Standard methods (Kikkawa et al., 1983; Roskoski, 1983), with some modifications (Tuominen et al., 1991), were used. Treatments were stopped by washing the cells with ice-cold Ca++-free medium (pH 7.5). Cells were disrupted by sonication. The suspension was centrifuged (100 000 x g, 60 min, 4°C), first without (soluble extract) and then with (particulate extract) 1 ml l-1 Triton X-100. In the assay, the protein extracts were allowed to catalyse histone (0.3 mg ml-1 calf thymus histone type III-S; Sigma) phosphorylation for 5 min at 30°C in the presence of adenosine g-32P-triphosphate (Amersham), both with and without the PKC activators: 1 mM CaCl2, 40 g ml-1 phosphatidylserine and 8 g ml-1 1,2-sn-dioctanoylglycerol. The reaction was terminated by spotting reaction mixture onto P81 phosphocellulose paper (Whatman, Maidstone, UK), and washing the papers in 75 mM phosphoric acid. The trapped radioactivity was counted after air-drying. PKC activity was calculated by subtracting the non-specific activity from total activity. Results are expressed as nmoles of inorganic phosphate incorporated into histone min-1 mg-1 of protein.
For short treatments, the growing medium was replaced by HEPES- and NaHCO3-buffered saline (pH 7.4 at 5% CO2), and the cells were allowed to stabilise for 60 min. [3H]Phorbol-12,13-dibutyrate (PDBu, Amersham) with (non-specific binding) or without (total binding) 1 M phorbol 12-myristate 13-acetate (PMA) was added into the wells. The 24-h experiments were run in the growing medium, and 15 min before stopping the treatment [3H]PDBu with or without PMA was added. The final concentration of [3H]PDBu was 10 nM. Treatments were terminated by washing the cells with isotonic saline. The bound radioactivity was measured, and results are expressed as specific binding.
Adult female Wistar rats (HsdBrlHan:WIST) with free access to food and water were used. Brains were obtained after decapitation, and brain areas were dissected on ice. The study protocol was approved by the University of Helsinki ethics committee for animal experiments.
Tissues and cells were homogenised in a glass-teflon homogeniser in hypotonic Tris buffer containing protease inhibitors. The homogenates were spun twice at 30 000 x g for 15 min, and the pellets were resuspended in fresh buffer. [3H]Epibatidine (NEN Life Science Products, Boston, MA, USA) was used as the nicotinic label. The experiments were performed in polypropene tubes in 50 mM Tris HCl buffer (pH 7.4) at 37°C. The binding time was 4 h. To determine non-specific binding, 300 M nicotine was added to the incubations. In saturation binding experiments, the [3H]epibatidine concentration was 14.5 pM - 6.75 nM (chromaffin cells) or 10 pM - 2.15 nM (brain). In competition experiments, single concentrations of 100 pM (chromaffin cells) and 250 pM (brain) were used. The incubations were terminated by using Brandell M-24R cell harvester. The bound ligand was trapped in Whatman GF/B filters presoaked for five hours in 5 mg ml-1 polyethyleneimine, and the filters were washed three times with cold Tris buffer. The filters were counted for radioactivity after overnight incubation in the scintillation liquid.
If not otherwise stated, the chemicals were obtained from standard commercial sources. Inorganic salts and solvents were of analysis grade and were supplied by Merck, Darmstadt, Germany and by J.T. Baker, Deventer, The Netherlands. The pharmacological agents and the cell culture media were mostly from Sigma, St. Louis, MO, USA. Other suppliers included Tocris Cookson, Bristol, UK; Alomone Labs, Jerusalem, Israel; and LC Services, Woburn, MA, USA.
Arithmetic means, standard deviations and standard errors of means were calculated from the numeric data. Parametric tests were preferred whenever data distribution allowed. Angular transformation for percentage data was done if required to homogenise the variances (IV). In comparing two groups with a dependent variable, paired t-test was used (V).
Primarily, analysis of variance was used for estimating the existence of differences. Repeated measures tests were used where appropriate (II, V). Post hoc tests for multiple comparisons were Tukey's test (II - IV) or Dunnett's test (II, V). For non-parametric analyses, the Kruskall-Wallis test was used to evaluate whether a difference existed between any groups. Non-parametric post hoc tests were done using Mann-Whitney U-test, followed by Dunn-Sidak (I) or Bonferroni (III) procedure.
For receptor binding data (VI), nonlinear regression analyses were used. The data were fitted to one-site and two-site models, and the fits were compared by F-test. The analyses were performed with Prism 2.0b for Macintosh (GraphPad Software, San Diego, CA, USA).