Vol. 4, No. 1, September 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 61-66 ENHANCEMENT BY F-ACTIN OF MGATP-DEPENDENT DOPAMINE UPTAKE INTO ISOLATED CHROMAFFIN GRANULES Kyoji Morita ~)*, Stuart M Tomares 2) and Harvey B. Pollard Laboratory of Cell Biology and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 2892 ~)Present address: Department of Pharmacology, Tokushima University School of Medicine, 3-15-18 Kuramoto, Tokushima 77, Japan "-)Present address: Pediatric Pulmonary Division, Johns Hopkins Hospital, Baltimore, Maryland 21287 Received March 16, 1996 SUMMARY Interaction of chromatfin granules with the microfilament network in the cytoplasmic space of adrenal medullary cells has been proposed, but the influence of chromaffin granule-microfilament interaction on granule function has not yet been studied. In the present study, the effect of F-actin on dopamine uptake into isolated chromaffin granules was examined. The MgATP-dependent component of dopamine uptake was significantly enhanced by F-actin. In contrast, catecholamine ettlux from the granules was not significantly altered by F-actin under the conditions in which the enhancement of MgATP-dependent dopamine uptake was observed. These findings suggest that the interaction of chromaffin granules with the microfilament network results in the enhancement of MgATP-dependent dopamine uptake into the granules, thus modulating the accumulation and storage of catecholamines in the adrenal medullary cell. Key words: Chromaffin granule, Dopamine uptake, F-Actin, Microfilament network INTRODUCTION Chromaffin granules are known to store various neuropeptides (enkephalins, neuropeptide Y) and proteins (dopamine 13-hydroxylase, chromogranins) as well as large quantities of catecholamines and ATP (1,2), and play an important role in catecholamine secretion as a part of the exocytotic machinery (3). In addition, since dopamine 13-hydroxylase, the enzyme catalyzing the conversion of dopamine to norepinephrine, is localized within chromaffin granules, the granules are generally * TO whom all correspondence should be addressed. 61 139-9712/96/161-655./ Copyright 1996 by Academic Press Australia. All rights of reproduction in any form reserved.
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL appreciated as the site of norepinephrine biosynthesis in the adrenal medullary cell (4). Thus, the transport of dopamine into chromaffin granules is thought to be a critical step in both the secretion and the biosynthesis of norepinephrine. Hence, catecholamine transport into chromaftin granules has been extensively studied and well characterized. Catecholamines are taken up into the isolated granules (5-8) and the granule ghosts (9-11) in a MgATP-dependent manner, and the mechanism by which catecholamines are accumulated within the granules has been fully defined (12,13). However, the regulatory mechanism of catecholamine transport into chromaffin granules has not yet been elucidated. Earlier studies have shown that F-actin interacts with chromaffm granule membranes prepared from bovine adrenal medulla under conditions that approximate the intracellular environment of resting cells, and the granule membrane-actin interaction is inhibited by raising the free calcium concentration (14). Thus, the interaction of chromaffin granules with actin filaments has been suggested to represent an aspect of the mechanism regulating the translocation of chromaffin granules in the chromaffin cell (15,16). Furthermore, the interaction of chromaftin granule membranes with F-actin has been shown to protect the membrane-bound ATPase against thermal inactivation as a consequence of stabilizing the granule membrane structure, suggesting a possible role of the microfilament network in the preservation of chromaffin granule function as well as the position of granules within the adrenal medullary cell (17). Since the transport of catecholamines into chromaftin granules is tightly linked to membrane-bound ATPase activity (12,13), it seems reasonable to assume that the microfilament network can modulate the transport of catecholamines into chromaffin granules within the cell. In the present study, the effect of F-actin on MgATP-dependent dopamine uptake into isolated chromaffin granules was examined. Dopamine uptake into the granules was significantly enhanced by the presence of F-actin, suggesting a putative role of microfilaments as a cytoplasmic factor regulating the transport of catecholamines into chromaftin granules in the adrenal medullary cell. MATERIALS AND METHODS Chromaffin granules were isolated from flesh bovine adrenal medulla by differential centdfugation (18), and suspended in buffered sucrose (.3 M sucrose - 2 mm PIPES, ph 6.8). The granule concentration was adjusted to an optical density of approximately 5. at 54 nm, and stored on ice until used. Actin was purified from an acetone powder of rabbit skeletal muscle according to the method of Spudich and Watt (19). Dopamine uptake into chromaffin granules was determined according to the method of Ramu et al. (8) with minor modifications. Briefly, the granule suspension (25 BI) was mixed with an equal volume of the incubation medium consisting of.3 M sucrose, 2 mm PIPES (ph 6.8) and 62
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 1 I~M [3H]dopamine (.25 ~tci) with or without 2 mm MgATP, and incubated at 37 C for different time periods. The reaction was stopped by adding 1 ml of ice-cold buffered sucrose, and the tubes were then centrifuged at 2, x g for 2 min at 4 C. The precipitated granules were lysed by adding 1 ml of ice-cold water followed by a freeze-thaw cycle, and proteins in the granule lysates were precipitated by adding 25 ~tl of 2 M perchloric acid. After centrifuging at 2, x g for 2 rain, radioactivity in the supernatant fraction was determined by liquid scintillation counter. The amount of [3H]dopamine taken up into the granules was calculated on the basis of the specific activity of [3H]dopamine in the incubation mixture, and MgATP-dependent uptake was calculated by subtracting the uptake obtained in the absence of MgATP from that obtained in the presence of MgATP. Catecholamine efflux from chromaffin granules was determined by measuring the amount of catecholamines released into the medium during incubation. The isolated granules were incubated with unlabeled dopamine as described above, and the reaction was stopped by chilling the tubes on ice. The granules were precipitated, and the resulting supernatant (4 ~tl) was acidified by adding 1 Ixl of glacial acetic acid. The amount of catecholamines in the supernatant were fluorometrically determined by the modified trihydroxyindote method (2,21). 3,4-[ring-2,5,6-3H]Dopamine was purchased from Du Pont-New England Nuclear. ATP and dopamine were obtained from Sigma Chemical Co. Other chemicals used were of commercially available reagent grade. RESULTS AND DISCUSSION The influence of microfilarnents on catecholamine transport into chromaffin granules was studied using a model system consisting of isolated bovine adrenal chromaffin granules and rabbit skeletal muscle F-actin. As shown in Fig. 1, MgATP-dependent dopamine uptake into the granules was increasing according to the incubation time, and then reached the maximum level at 3 rain. The MgATP-dependent uptake was significantly enhanced by incubating the granules with F-actin for 7.5 and 15 min, but the significant effect of F-actin on the uptake was not observed after 3 min. In contrast, MgATP-dependent dopamine uptake was not affected by the addition of bovine serum albumin (Fig. 2). These results seem to indicate that the interaction of chromaffin granules with microfilaments results in an increase in the rate of dopamine uptake, but not the maximum uptake, into the granules under the conditions used here, and furthermore suggest that this enhancement of dopamine uptake may reflect a specific action of F-actin on the granule membranes. In the previous study, MgATP has been proposed to play several independent roles in the flux of catecholamines across the chromaffin granule membranes (8). In addition to the unidirectional uptake of catecholamines into the granules, MgATP has been suggested to catalyze the exchange of catecholamines across the granule membranes through a mechanism dependent on ATPase activity and to prevent the spontaneous leakage of catecholamines from the granules in a manner 63
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 3 [~ : F-actin ( - ) i : F-actin ( + ) E._c 2 a Q.. a "B E 1 C 7.5 15 3 45 Time ( rain ) Fig. 1. Effect of F-actin on MgATP-dependent dopamine uptake into chromaffin granules. Isolated granules were incubated with or without F-actin (25 ~tg/ml) at 37 C for various time periods in the mixture containing [3H]dopamine, and the MgATP-dependent uptake was then determined as described in the text. Values are the mean + S.E. (n = 6, *P<.1). 3 [-'-'l : BSA(-) l: BSA (*) E Q. O ~ 2 -o _~ =E "" O Q., & 1 7.5 15 3 45 Time ( min ) Fig. 2. Effect of BSA on MgATP-dependent dopamine uptake into chromaffin granules. Isolated granules were incubated with or without BSA (4 mg/ml) at 37 C for various time periods in the mixture containing [3H]dopamine, and the MgATP-dependent uptake was then determined as described in the text. Values are the mean + S.E. (n = 6). 64
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL r.. =,- 8 '5 15 1 : F-actin ( - ) : F-actin ( + ) X LU ~D O3 o 5, l, I, I 15 3 45 Time ( rain ) Fig. 3. Effect of F-actin on spontaneous catecholamine efflux from chromaffin granules. Isolated granules were incubated with or without F-actin (25 ~tg/ml) at 37 C for various time periods in the mixture containing dopamine and MgATP, and eatecholamines released into the medium was then determined as described in the text. Values are the mean + S.E (n = 6). independent of this enzyme activity. It is therefore still questionable whether the enhancement of MgATP-dependent dopamine uptake by F-actin is actually due to the facilitation of dopamine transport into the granules. To address this question, the isolated granules were incubated with or without F-actin in the medium containing dopamine and MgATP, and catecholamine efflux from the granules was determined. As shown in Fig. 3, a rapid efflux of catecholamines was observed during the incubation, and the efflux was not affected by the presence of F-actin under conditions in which the enhancement of MgATP-dependent dopamine uptake by F-actin was observed. It is therefore conceivable that F-actin may enhance MgATP-dependent dopamine uptake into isolated chromaffin granules as a consequence of facilitating the unidirectional uptake across the granule membranes rather than preventing the efflux of radioactive dopamine taken up into the granules. Thus, the present study provide the first evidence that the interaction of chromaffin granules with the microfilament network may play a role in the regulation of catecholamine transport into the granules in the adrenal medullary cell. 65
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