Adrenal chromaffin cells are useful for studying the mechanism of stimulus-secretion coupling, and are regarded as a model for catecholamine-containing neurons. Physiological stimulation of the cells causes increases in the levels of intracellular free Ca2+ ([Ca2+]i) from both the intracellular pool and extracellular spaces (1). The increase in [Ca2+]i leads to the stimulation of catecholamine release (2,3) and activation of its biosynthesis (4). Tyrosine hydroxylase catalyzes the rate-limiting step in the biosynthesis of catecholamine (5). In bovine adrenal chromaffin cells, this enzyme is phosphorylated and activated by an increase in cylic AMP (cAMP) (6) or elevation [Ca2+]i in the cells (7).
The cardiotonic glycoside ouabain, an inhibitor of Na+/K+-ATPase, increases the force of contraction of the heart through a Ca2+-dependent mechanism(s). Therefore, the cardiotonic glycosides are used for treatment of heart failure. Although the short-term effect of digitalis glycoside on catecholamine secretion has been reported (8), little is known about the long-term effects of ouabain on the sympathetic nervous system, which regulates contraction of the heart.
In the present study, to determine whether long-term treatment of ouabain activates tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, we examined the long-term effects of ouabain on catecholamine formation in cultured bovine adrenal chromaffin cells.
Cell preparation and culture
Bovine adrenal chromaffin cells were dispersed enzymatically as described previously (1). Briefly, the medulla was sliced with a hand slicer, and the slices were digested in medium containing 0.1% collagenase, 0.01% soybean trypsin inhibitor and 0.5% bovine serum albumin in balanced salt solution (135 mM NaCl, 5.6 mM KCl, 1.2 mM MgSO4, 2.2 mM CaCl2, 10 mM glucose and 20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid/NaOH; pH 7.4). Cells were plated in 35 mm culture dishes at a density of 2 × 106 cells/dish. They were maintained for 3-5 days as monolayer cultures in Eagle's basal medium supplemented with 5% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamycin, 2.5 μg/ml fungizone and 10 μM cytosine arabinoside. The cells were then incubated with the culture medium containing various concentrations of ouabain for 0-48 h.
Measurement of catecholamine formation
The cells were washed with 1 ml balanced salt solution and incubated at 37°C for 30 min in 1 ml balanced salt solution (BSS) containing 20 μM L-[14C]tyrosine (0.5 μCi). The medium was discarded, and the cells were washed twice with 1 ml ice-cold balanced salt solution on ice, and lysed by adding 1 ml of 0.4 N perchloric acid followed by one cycle of freeze-thaw. Radioactivity in the acid extract was counted in a liquid scintillation spectrometer to determine the amount of [14C]tyrosine taken up into the cells. [14C]Catecholamines in the acid extract were isolated on aluminum hydroxide, and radioactivity eluted from the gel was counted by liquid scintillation spectrometry (9). In some experiments, L-[14C]DOPA (50 μM, 0.5 μCi) was used as the substrate instead of L-[14C]tyrosine. [14C]Catecholamines formed from [14C]-labeled substrates were measured by ion-exchange chromatography on a Duolite C-25 column (9).
Figure 1A shows the formation of [14C]catecholamines from [14C]tyrosine on adrenal chromaffin cells cultured with various concentrations of ouabain. Ouabain treatment stimulated [14C]catecholamine formation dose-dependently at concentrations of 10-300 nM. Ouabain increased [14C]catecholamine formation significantly at 30 nM and had a maximal effect at 300 nM. This stimulatory effect of ouabain was not due to an increase in uptake of [14C]tyrosine into the cells because the total radioactivity within the cells did not change by ouabain treatment. Moreover, it did not stimulate [14C]catecholamine formation with [14C]DOPA instead of [14C]tyrosine as a substrate (control, 12.5 ± 1.2 nmol/dish; 100 nM ouabain for 24 h treated cells, 10.1 ± 1.1 nmol/dish), suggesting that ouabain treatment increased the conversion of tyrosine to DOPA catalyzed by tyrosine hydroxylase, the rate-limiting step in catecholamine biosynthesis. The increase in [14C]catecholamine formation was dependent on the period of ouabain treatment. Ouabain treatment for 8 h showed the maximal effect on [14C]catecholamine formation (Fig. 1B).
To determine whether ouabain-induced [14C]catecholamine formation was mediated by calcium-dependent mechanism(s), the cells were treated with various concentrations of extracellular Ca2+. As shown in Fig. 2, [14C]catecholamine formation stimulated by ouabain was dependent on extracellular Ca2+ concentrations.
The formation of catecholamines is regulated by activation and/or induction of tyrosine hydroxylase (10,11). We determined whether the effect of ouabain on catecholamine formation was mediated by the activation or induction of tyrosine hydroxylase using the protein synthesis inhibitor cycloheximide or the RNA synthesis inhibitor actinomycin D. As shown in Fig. 3, treatment of cycloheximide or actinomycin D inhibited [14C]catecholamine formation induced by dibutyl cAMP or carbachol, but not by ouabain. These observations suggested that ouabain-induced [14C]catecholamine formation is regulated by activation of tyrosine hydroxylase in adrenal chromaffin cells.
In the present study, we examined, for the first time, the mechanism(s) by which long-term treatment with ouabain stimulates catecholamine formation in cultured bovine adrenal chromaffin cells. Ouabain treatment increased [14C]catecholamine formation from [14C]tyrosine (but not from [14C]DOPA) in a concentration-dependent manner, suggesting that ouabain increases the conversion of tyrosine to DOPA, the rate-limiting step in the biosynthesis of catecholamines.
The activation or induction of tyrosine hydroxylase by increases in [Ca2+]i is thought to be mediated by calcium-dependent protein kinases (calcium/calmodulin-dependent protein kinase and protein kinase C) in a number of in vitro and in situ systems (7,12,13). Ouabain decreases the activity of Na+/K+-ATPase in several tissues including adrenal chromaffin cells (14). Inhibition of Na+/K+-ATPase activity elevates the level of intracellular Na+, leading to stimulation of Ca2+ influx in adrenal chromaffin cells (8). In the present study, the stimulatory effect of ouabain on catecholamine formation was shown to be dependent on extracellular Ca2+ concentration (Fig. 2) and was not inhibited by cycloheximide, an inhibitor of protein synthesis (15), or actinomycin D, an inhibitor of RNA synthesis (16)(Fig. 3). Thus, long-term treatment with ouabain activates tyrosine hydroxylase through calcium-dependent mechanism(s).
The present results suggested that ouabain, an inhibitor of Na+/K+-ATPase, shows continuous activation of hydroxylation of tyrosine in cultured bovine adrenal chromaffin cells. Therefore, cardiotonic glycoside ouabain may influence not only cardiac myocytes, but also the sympathetic nervous system, resulting in augmentation of the contractility of the heart.
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The symposium and the publication of this supplement were supported by an educational grant from Novartis Pharma K.K. Tokyo, Japan.