Catecholamine Transport by Isolated Chromaffin Granules

Transcription

1 THE JOURNAL OF BlOLOClCAL CHEMISTRY Vol. 256, No 3, Issue of February 1, pp , 1981 Printed in U. S A. Catecholamine Transport by Isolated Chromaffin Granules INFLUENCE OF MgATP AND A DLSULFONIC STILBENE ON (R)-NOREPINEPHRINE/EPINEPHRINE EXCHANGE AND SPONTANEOUS EPINEPHRINE EFFLUX* (Received for publication, July 1, 1979, and in revised form, June 24, 198) Avner Ramu+g, Christopher J. Pazolest, Carl E. Creutzl, and Harvey B. Pollard1 From the *Reproduction Research Branch, National Institute of Child Health and Human Deuelopment, National Institutes of Health, Bethesda, Maryland 225 and the!clinical Hematology Branch, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Znsitutes of Health, Bethesda, Maryland 225 Isolated chromaffin granules were found to accumulate exogenous (R)-[3H]norepinephrine in the presence of Mg2+-ATP by exchange for endogenous epinephrine at nearly a 1:1 ratio. At low concentrations of (R)- norepinephrine (t2 PM), uptake followed Michaelis- Menten kinetics with regard to both MgATP and [@)- norepinephrine] (K, = 123 PM and 49 PM, respectively). At high concentrations of (R)-norepinephrine (e.g. 5.1 mm), substantial amounts of additional (R)-[3H]norepinephrine entered granules in a manner independent of [MgATP]. Incubation of granules without added MgATP or external (R)-norepinephrine resulted in the release of substantial amounts of endogenous epinephrine into the medium, and addition of ATP suppressed this epinephrine leakage by a saturable mechanism (K, = 4 p ~). However, while the MgATP-dependent (R)- [3H]norepinephrine/epinephrine exchange and ATPase activity were both inhibited by 4-acetamido-4 4sothiocyanostilbene-2,2 -disulfonic acid (SITS), SITS did not affect either ATP-inhibited epinephrine leakage or ATP-independent (R)-norepinephrine uptake. We conclude from these data that ATP plays several independent roles in the function of intact granules. One role of ATP is to catalyze bidirectional catecholamine flux across the granule membrane rather than unidirectional net accumulation into intact granules. This flux depends on the ATPase activity. A second role, apparently independent of the ATPase activity, is to prevent spontaneous epinephrine leakage from granules. The fact that ATP is required for an otherwise isoenergetic exchange of catecholamines has led us to certain general conclusions about transport mechanism in this system, as well as a consideration of the possible relevance of the heterogeneous structure of the granule interior to transport properties. Isolated chromaffin granules take up (R)-norepinephrine and other catecholamines in the presence of Mg +-ATP (l), and the process has attracted recent attention both as a prototype for catecholamine transport in general (2,3) and as a possible specific process in chromaffin granule assembly (4, 5). At low concentrations of free catecholamines, transport requires ATP hydrolysis. For example, both Taugner and Hasselbach (6) and Ferris et al. (7) showed that the ATPase * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 Present address, Department of Pharmacology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel inhibitor N-ethylmaleimide blocked catecholamine uptake. In addition, Hoffman et al. (8) and Phillips (9) reported that ATP analogues that were not substrates for the ATPase (such as AppNHp, AppCHp, or ApCHepp) did not support uptake of (R)-[ H]norepinephne. However, the mechanism by which ATP hydrolysis could induce uptake of exogenous catecholamines in the face of a tremendous internal catecholamine concentration (approximately.5 M) has attracted much continuing speculation. Radda and his colleagues (1-12) found that ATP-dependent uptake by granules was inhibited by proton ionophores, and they proposed that catecholamines entered granules via a carrier in exchange for a proton previously pumped in by the membrane ATPase. Johnson and Scarpa (3) have proposed, as an alternative, that catecholamines might equilibrate with the naturally occurring ph gradient across the granule membrane and that the ATPase might act to maintain this gradient by the proton-pumping ATPase mechanism. These models were all constructed on the assumption that the ultimate function of the ATP-mediated uptake system in intact granules was to catalyze net accumulation of catecholamines. In the present studies we attempted to test this assumption directly and report that under optimal transport conditions at 37 C, uptake of (R)-[ Hlnorepinephrine is closely balanced by release of endogenous epinephrine in a nearly 1:1 molecular ratio. Therefore, ATP appears catalyze to exchange of catecholamines rather than net accumulation in intact granules. As a separate effect, we also found that ATP suppressed spontaneous epinephrine release from granules, an observation consistent with earlier reports of Kirshner (13) and Slotkin and Green (14). We conclude that the final catecholamine content of the granule core must be precisely maintained and that the ATP requirement for what is essentially an exchange event suggests that the catecholamine transport mechanism may be more complex than previously anticipated. EXPERIMENTAL PROCEDURES Isolation of Chromaffin Granules-Bovine adrenal glands were obtained at a local slaughterhouse and transferred on ice within 2 h of killing to the laboratory. Chromaffin granules were purified from a homogenate of adrenal medulla by differential centrifugation as described by Pollard et al. (15) and Pazoles and Pollard (IS). This preparation contained 1.85?.21 pmol of epinephrine/mg of protein, and (Rknorepinephrine was less than 58 of the total catecholamines (norepinephrine plus epinephrine). Analysis of Catecholamine Transport-Granules in.25 ml of.3 M sucrose were mixed with 3.25 ml of an incubation medium consisting of 2 mm KCl, 5 mm 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid-naoh buffer, ph 7.5, various concentrations of MgSOj and NaZATP (1:l molar ratio), (R)-[ Hlnorepinephrine-HC1 and (R)-norepinephrine bitartrate in different proportions to yield constant ra-

2 123 ATP-activated Catecholamine Exchange dioactivity and sufficient sucrose to make the solution isotonic with 335 milliosmoles/liter. The incubation medium was prewarmed at 37 C for 1 min prior to the addition of the granules. The granule suspension was adjusted to an optical density of 5. at 54 nm and stored on ice until use. This granule mixture contained.43 f.8 (n = 3) mg of protein in a.25-ml aliquot. The reaction was stopped by adding 2 ml of ice-cold.33 M sucrose, and the tubes were immediately transferred to an ice bath. Then the tubes were centrifuged at 2, X g for 3 min at 2-4 C. Supernatants were reserved for later analysis, and the pellets were washed by filling the plastic centrifuge tube with ice-cold.33 M sucrose taking care not to dislodge the pellet. After decanting the supernatant, the pellets were disrupted by adding 1 ml of distilled water and freezing and thawing. The thawed pellets were homogenized by vigorous agitation on a Vortex mixer, and.2 ml of the suspension as well as of the supernatant was mixed with 12 ml of Aquasol forradiometric determination of 'H. Protein and catecholamine concentrations were determined on all pellet suspensions and supernatant solutions. Chemical and ESnzymatic Analysis-Protein was measured by the Bradford method, as described for application to adrenal medulla tissue (17). Epinephrine and norepinephrine were measured by the trihydroxy indole reaction, as described by Anton and Sayre (18). ATPase activity was measured by the colorimetric method of Itaya and Vi (19). RESULTS by Chromaffin Granules measured as a function of MgATP concentration at a constant (R)-norepinephrine concentration of 57 p ~ and, the rates were found to be constant for at least 2 min (Fig. 2.4). The rate of uptake was saturable with respect to MgATP concentration, and kinetic constants were K,, = 123 ~ L M and V,,,,, = 8.14 nmol of (R)-norepinephrine/(mg of protein X min). Hydrolysis of ATP in each condition at the end of 2 min was estimated from concurrent ATPase assays be to 12.9% (.2 mm MgATP); 7.% (.5 mm MgATP); 4.% (1. mm MgATP); and.8% (5 mm MgATP). The actual ATP concentration in the medium in the absence of exogenous ATP was calculated to be at most 3.5,UM at the beginning of the incubation and 11.1 p~ after 2 min at 37 C. These calculations were based on the free epi- nephrine concentration measured and a 451 ratio of catecholamine to ATP in granules and granule lysate (26). MgATP-independent Uptake of (R)- f3h]norepinephrine-it has been appreciated for many years that (R,S)-norepinephrine in high concentration can be taken by up granules in a temperature-sensitive but ATP-independent fashion (27). As shown in Table I, we were able to duplicate this finding with (R)-norepinephrine. Temperature-dependent uptake at a high (R)-norepinephrine concentration (5.7 mm) in the absence of ATP (Table I, line 3) was 37% of that in its Dependence of (R)- f 3H]Norepinephrine Uptake on (R)- presence (Table I, line 4). By contrast, with 57 ~ L M (R)-nor- Norepinephrine Concentration-Kinetic studies on (R)-nor- epinephrine, such uptake without ATP (Table I, line 7) was epinephrine uptake by isolated granules have been performed in the past. However, Jonasson et al. (2) and Lundborg (21) studied (Rbnorepinephrine uptake as a function of only very only 5% of that with ATP (Table I, line 8). Obviously, ATPdependent uptake was observed with both high and low (R)- norepinephrine concentrations, but ATP-independent uptake high (R)-norepinephrine concentrations, and the ATP re- was primarily observed at high concentrations of (R)-norepiquirement was studied by Kirshner (1, 13) and Carlsson et al. nephrine. As indicated in Fig. 2, 1. and.5 mm MgATP were (22), under conditions where hydrolysis of ATP was estimated almost equally effective at supporting uptake in the presence in one case to be as high as 9% of the total. It was thus necessary to characterize the bovine granule catecholamine uptake system with respect to both (R)-norepinephrine and ATP concentrations before investigating the nature of the transport process. The uptake of (R)-["Hlnorepinephrine in the presence of 1 mm MgATP was linear for more than 2 min over a wide range of (R)-norepinephrine concentration (Fig. la). The uptake rates were plotted as a function of (R)-norepinephrine " concentration (Fig. lb), and the Lineweaver-Burk plot (Fig. O 1 M loo 15 1B, inset) proved linear. The K, for (R)-norepinephrine was TIME, MIN [/-NE]. PM p ~ and, the V,,, was 11.8 nmol of (R)-norepinephrine/ FIG. 1. Kinetics of (R)-[3H]norepinephrine uptake as a func- (mg of protein X min). Over the course of these experiments, tion of external (R)-norepinephrine (I-NE) concentration. A, the values for these constants varied by no more than 5%. Our time courses of uptake at different (R)-norepinephrine concentra- K,,, value was compatible with similar values for intact rat (23) tions: a = 14.3 pm; b = 28.6 pm; c = 57.2 pm; d = pm; e = and rabbit (24) granules and bovine granule membranes (9, p ~ E,. substrate-velocity plots of data in A. Correlation coefficients were: 14.3 p~ (r =.979); 28.6 p~ (r =.977); 57.2 p~ (r =.993); 25). On the basis of ATPase measurement, we estimated that p~ (r =.996); p~ (r =.996). The inset is a Lineweaverno more than 4% of the added ATP was hydrolyzed by the Burk plot of data in E. end of 2 min. (R)Norepinephrine also remained intact over this time period as defined by the specific fluorometric assay (data not shown). Stereospecificity of (R)-PHINorepinephrine Uptake-Uptake of norepinephrine was highly stereospecific for the (R)- form at low substrate concentrations. The relative transport of (R)-[,"H]norepinephrine and (R,S)-["Hlnorepinephrine was compared in a reaction mixture containing 57 p~ (Rbnorepinephrine and 1 pci of either (R)-["Hlnorepinephrine or (R,S)- [,"H]norepinephrine and 1 mm MgATP. The rate of "H-uptake into chromaffin granules incubated with (R,S)-["Hlnorepi- TIME, MIN. [MQATPI. mm nephrine (15,8 cpm/(mg of protein X rnin)) was half the rate oph-uptake in granules incubated with (R)-["H]norepi- FIG. 2. Kinetics of (R)-['Hlnorepinephrine uptake as a funcnephrine (32,752 cpm/(mg of protein x min)). We concluded tion of external M&'-ATP concentration. A, time courses of uptake at differentmg"-atpconcentrations: a = no exogenous that at low concentrations of norepinephrine, the (R)-form Mg"'-ATP (r =.975); b = 5 PM (r =.984); c = 1 p~ (r =.998); was transported preferentially. d = 2 PM (r =.999); e = 5 p~ (r =.999); f= 1 mm (r =.998). Dependence of (R)-i3HjNorepinephrine Uptake on E, substrate velocity plots of data in A. The inset is a Lineweaver- MgATP-The uptake of (R)-[,"H]norepinephrine was also Burk plot of the data in B. 1-NE, (R)-norepinephrine.

3 TIME, ATP-activated Catecholamine Exchange by Chromaffin Granules 1231 of 57 PM (R)-norepinephrine, thus demonstrating that the use these data that ATP-dependent uptake of (R)-[ Hlnorepiof different ATP concentrations in the experiments described nephrine by granules probably involved an exchange for enin Table I had no effect on the relative uptake rates in the dogenous epinephrine on a 1:l basis. two experiments. In further support of this concept, we also found that in the Exchange of Transported (R)- Norepinephrine for Endog- presence of 1 mm MgATP but in the absence of added (R)- enous Epinephrine-Several recent studies on catecholamine norepinephrine, no change in the concentration of free epitransport by chromaffin granules (1-12, 28) have dealt with nephrine in the medium was observed for as long as 4 min possible mechanisms by which activity of the membrane (Fig. 3B). MgATPase could induce net accumulation of catecholamines. Influence of ATP Concentration on (R)-Norepinephrine/ To test this assumption about net accumulation, we measured Epinephrine Exchange-While pursuing the previous exper- (R)-norepinephrine uptake and endogenous epinephrine ef- iment, we discovered that incubation of granules in low conflux concurrently under various transport conditions. As centrations of MgATP led to much more epinephrine efflux shown in Fig. 3, both uptake and efflux, in 1 mm MgZ+-ATP, than (R)-norepinephrine uptake, and over the entire range of proceeded at linear rates proportional to the concentration of added (R)-norepinephrine. Thus, simple net accumulation of (R)-norepinephrine by granules appeared to be excluded. The data in Fig. 3 consisted of averages of duplicate deter- minations of both (R)-norepinephrine uptake and efflux at different times. It was thus apparent that if each individual uptake value were graphed as a function of the individual efflux values in a given tube, the possible correlation between the two processes might be measured. This was done in Fig. 4 in which a clear correlation between (R)-norepinephrine uptake and epinephrine release was demonstrated with a slope of.79 and a correlation coefficient of.887. This slope was not statistically different from 1. on the basis of Student s t test. This experiment was performed a second time in which the concentration of (R)-norepinephrine was varied from 12 to 571 PM with the MgATP concentration again maintained at 1 mm. In this case, the slope was.86 (r =.926) and again was not significantly different from 1.. We concluded from TABLE I The influence of temperature, MgATP, and (R)-norepinephrine concentration on uptake of (R)-norepinephrine (RbNorepinephrine PM a. 57 MgATP mm Tempera- ture (E)-Norepinephrine uptake Total c Nanomoles of (R)-norepinephrine/(min X mg of protein); total incubation time = 1 min. 12 I 7 16, lb e, ~ MIN TIME, I ~ FIG. 3. Influence of (R)-norepinephrine (1-NE) concentration on (R)-[3H]norepinephrine uptake and epinephrine release from granules in the presence of 1 mm M&+-ATP. A, time course of uptake at different (R)-norepinephrine concentrations: a = 14.3 pm; b = 28.6 p ~ c ; = 57.2 pm; d = p ~ e ; = PM. B, time course of epinephrine release at different (R)-norepinephrine concentrations. The letters represent the same experimental conditions as in A and come from the same experiments. MgATP concentrations (1 to 1 PM), no meaningful correlation between (R)-norepinephrine uptake and epinephrine efflux could be drawn. In a more direct analysis, we found that at higher MgATP concentrations, (R)-norepinephrine uptake just balanced epinephrine efflux (Fig. 5A). This could be due either to activation of catecholamine uptake (i.e. reuptake of epinephrine), or inhibition to of epinephrine efflux by MgATP. However, addition of MgATP to a granule suspension never reduced the amount of free epinephrine already present in the medium at zero time in our experiments (Fig. 3B), indicating that activation of the uptake system did not explain this effect of MgATP. Furthermore, much more epinephrine was released in the absence of MgATP than could have been reaccumulated by the uptake system in the presence of 1 mm Mg -ATP. For example, after 2 min of incubation without MgATP an increment of nmol of epinephrine was released into the medium (Fig. 5A, curue a). The uptake system could reaccumulate at most 4 nmol of this released epinephrine in 2 min if ATP were present, leading us to expect an increase over zero time levels of at least 15.4 nmol of free epinephrine. Instead, no increases in epinephrine levels in the medium were observed in the presence of 1 mm MgATP without added (R)-norepinephrine(Fig. 3B, curue no NE ). Therefore, the reaccumulation hypothesis cannot explain the suppression of epinephrine efflux by ATP. We also found that MgATP-induced suppression of spontaneous epinephrine efflux was a saturable function of the [MgATP] with an apparent K, of about 4 p~ (Fig. 5B). This indicated to us that the ATP-dependent suppression of epinephrine efflux was related to interaction of ATP with a specific granule site, distinct from that involved in the catecholamine exchange and possibly not mediated through ATPase activity. We realized that this hypothesis could be tested further ifwe were able to inhibit the ATPase without inhibiting the ability of ATP to block this epinephrine efflux process. Influence of SITS on (R)-Norepinephrine Uptake and ATPase Activity-The chromaffin granule ATPase is not sensitive to such well known ATPase inhibitors as ouabain or The value of 4 nmol of possible maximal reaccumulation of released epinephrine over 2 min in the presence of 1 mm Mg -ATP was computed as follows. The maximum amount of epinephrine found in the medium in the absence of ATP after 2 min was 2 nmol/ tube, corresponding to an epinephrine concentration of 57 p~ (Fig. 5A, curue a). From Fig. 3A (curue c) we can see that this concentration of catecholamine in the presence of 1 mm Mg -ATP would support the uptake of 4 nmol/pellet over a 2-min period. Since epinephrine and UWnorepinephrine are handled similarly by the granule uptake system (1, 14), we can calculate epinephrine uptake from our data on (R)-norepinephrine uptake. The free epinephrine concentration of 57 p ~ reached, after 2 min in the absence of added Mg -ATP is probably a very high estimate, and the true average value probably lies between this number and the initial free epinephrine concentra- tion (15.8 p ~). This would support an even lower uptake rate than 4 nmol/pellet/2 min. The abbreviation used is: SITS, 4-acetamido-4 -isothiocyano-stilbene-2.2 disulfonic acid.

4 1232 A TP-activated Catecholamine Exchange by Chromaffin Granules I- sbpe ~.79 z r =.887 [Mg-ATP] = 1 mm was also reported by others (7). These data suggested that the influence of SITS on (R)-norepinephrine uptake could be traced to its effect on the ATPase activity. SITS was also found to inhibit only ATP-dependent uptake of (R)-norepinephrine under conditions of high (R)-norepinephrine concentration. As indicated in Table 11, when [(R)-norepinephrine] was 5.7 mm, SITS did not affect the amount taken up at 4 C and reduced the ATP-dependent increment at 37 C to levels found in the absence of added ATP (cf. Table I, Eine 3) /-NE UPTAKE (nrnol/pellet~ FIG. 4. Correlation between epinephrine released and (R)- [3H]norepinephrine taken up at different (R)-norepinephrine (1-NE) concentrations between 143 and 229 CM. The Mg +-ATP concentration was held constant at 1 mm, and the incubation time was 1 to 4 min. The slope and correlation coefficients are shown in the figure. I / concentration was 57 p ~ and, the SITS concentrations included a = M ~ no SITS added b = 5 p ~ c ; = 5 p ~ and ; d = 5 p ~ B,. Lineweaver- TIME, MIN [Mg-ArP], mm Burk plot of data in A. The different SITS concentrations include a = no added SITS; b = 5 p ~ and ; c = 5 p ~. FIG. 5. Influence of M&+-ATP on spontaneous epinephrine (E) efflux (total epinephrine released minus epinephrine released in exchange for (R)-norepinephrine (I-NE) taken up). A, TABLE I1 time course of epinephrine efflux was measured in the presence of The effect of SITS and temperature on (R)-norepinephrine uptake various Mg -ATP concentrations and a constant exogenous (R)- at high exogenous (R)-norepinephrine concentration norepinephrine concentration of 57 PM. The various ATP concentra- Incubation was for a total of 1 min in the presence of 5 mm tions included: a = no exogenous Me-ATP; b = 5 p ~ c ; = 1 p ~ ; MgATP and 5.7 mm (R)-norepinephrine. Uptake is expressed as d = 5 p ~ Mg -ATP. concentrations of 1. and 2.5 mm gave curves nanomoles of (R)-norepinephrine/(min X mg of protein). quite similar to d and are not shown. B, the fraction of epinephrine (R)-Norepinephrine uptake efflux inhibited by Mg -ATP in A is graphed as a function of Mgz+- SITS Temperature ATP. Temperature de- Total pendent oligomycin. However, preliminary studies on the influence of stilbene disulfonates such as SITS led us to consider this and related compounds as possible inhibitors. SITS is one example of a class of aromatic, anionic, and impermeant 7.19 drugs which block anion transport in red cells (29) and isolated chromaffin granules (16). In the present work we were able to identify other actions of this drug including inhibition of ATP-dependent uptake of (R)-[ HI-norepinephrine(Fig. 6A and Table 11). On the basis of Lineweaver-Burk analysis (Fig. 6B), the inhibition proved to be of mixed character with an apparent inhibition constant for SITS of 19 /AM. In comparison, a similar study on reserpine inhibition of uptake yielded uncompetitive kinetics with respect to [MgATP] (data not shown). These data led us to investigate the possible action of SITS on ATPase activity. Granule ATPase activity under optimal uptake conditions was found to be linear for more than 2 min; and the kinetic constants, estimated from 1-min points, were found to be K,,, (ATP) = 145 PM and V,,, = 39.4 nmol of phosphate/(mg of protein X min) (Fig. 7). A similar V,,, value was obtained by others (3). Furthermore, as shown in Fig. 7A, SITS inhibited the granule ATPase; and according to Lineweaver-Burk analysis, the mechanism of inhibition was again mixed relative to [MgATP] with a K, = 58 PM (Fig. 7B). By contrast, reserpine had no effect on ATPase activity, as [Mg ATP] - 1 IS1 FIG. 6. Influence of SITS on the uptake of (R)-[3H]norepinephrine by chromaffin granules. A, substrate-velocity plot of (R)-norepinephrine (l-ne) uptake as a function of Mg +-ATP concentration at different SITS concentrations. The (R)-norepinephrine PM C r A I A boi [Mg -ATP]. mm FIG. 7. Influence of SITS on ATPase activity of chromaffin granules. A, substrate-velocity plot of ATPase activity as a function of Mg -ATP concentration in the presence of different amounts of SITS. SITS concentrations included: a = no added SITS; b = 1 FM; c = 5 p ~ and ; d = 1 p ~ B,. Lineweaver-Burk plot of data in A. The different letters also stand for the same SITS concentrations as in A.

5 ATP-activated Catecholamine TABLE I11 Influence of MgATP and SITS on (RJ-norepinephrine uptake and efflux of endogenous epinephrine at low exogenous (R)- norepinephrine concentration Incubations were for 1 min at 37 C in the presence of 57 PM (R)- norepinephrine except where indicated. rnm CLM nrnol nrnol nmol ()' Total amount of epinephrine measured in supernatant at end of incubation. Represents epinephrine released beyond that exchanged for (R)- norepinephrine; calculated by subtracting value for (R)-norepinephrine uptake from that for total free epinephrine. No (R)-norepinephrine was added, and the amount of free epinephrine at zero time was 33. nmol. Exchange by Chromaffin Granules 1233 electrical potential (34), or organization of the granule interior. In view of these properties of SITS, we went on to evaluate At present we have no evidence for or against direct influences the effect of SITS on ATP-suppressed spontaneous epinephof ATP on the carrier, except for the finding that ATP did rine efflux. Granules were incubated in 1 mm MgATP and not affect the affinity of the transport system for (R)-norepivarious concentrations of SITS up to 5 PM. As shown in nephrine. Table 111, granules in media lacking MgATP released sub- The granule interior consists of an osmotically inert core stantial amounts of epinephrine (line I) while granules incu- (containing protein, catecholamine, ATP, and other compobated in media containing 1 mm MgATP released no net epinephrine over that found at zero time (line 2). However, nents) and an intragranular aqueous phase (4.35 pl/mg of when the granules in 1 mm MgATP were also allowed to protein) (15, 35). Since the core may supply at least part of accumulate 25 nmol of (R)-norepinephrine, then an equivalent the epinephrine that is eventually exchanged for exogenous amount of epinephrine ( = 19.6 nmol) was released (R)-norepinephrine, it is possible that this released epinephinto the medium (line 3). Thus this system was well coupled rine necessarily passes through the aqueous phase during the in terms of (R)-norepinephrine/epinephrine exchange and was suitable for analysis of SITS effects on the exchange process. In the presence of SITS, ATP-induced uptake of (R)-[:'H]- norepinephrine was inhibited and the total amount of free epinephrine reduced. However, upon correcting for catecholamine exchange by subtracting the amount of (R)-norepinephrine taken up from the total free epinephrine, it was found that SITS did not affect ATP suppression of epinephrine efflux. This suggested the ATP suppression of epinephrine efflux was indeed independent of ATPase activity since the latter was sensitive to SITS. Influence of ATP on K,,, for (R)-Norepinephrine Uptake by Granules-We considered the possibility that the alternative site of action of ATP on the transport system might be on the putative (R)-norepinephrine carrier itself. We subjected this hypothesis to a partial test by asking if MgATP (over the range of 12.5 PM to 1 mm) affected the binding constant (K,) for (R)-norepinephrine in the uptake process. The result was that the apparent K,,, for (R)-norepinephrine was essentially unaffected by MgATP concentration. We concluded that if ATP affected the carrier directly, it did not do so by modifying the affinity of the carrier for its substrate. DISCUSSION The main conclusion drawn from this study of ($?)-["HInorepinephrine uptake by isolated chromaffin granules is that the process, under optimal conditions, involves an exchange of exogenous (R)-norepinephrine for endogenous epinephrine. At suboptimal ATP concentrations more epinephrine is released than is (R)-norepinephrine accumulated, demonstrat- ing that mature granules can exist in a depleted state but are incapable of net increment in catecholamine content. This is an unexpected result in view of the requirement for MgATP in the process and the likely involvement of the membrane ATPase. Indeed, many previously published analyses of catecholamine uptake in intact granules have concerned themselves with mechanisms for presumed net accumulation of substrate (e.g. Refs ). There are several mechanistic consequences of these exchange data that can be profitably considered if we presume that the cytoplasmic concentration of ATP in chromaffin cells is at least 1 mm. One indication from these data is that the exchange process is likely to occur via a membrane carrier system, since the kinetics and the preference of the system for the (R)-isomer of norepinephrine are consistent with this concept. Pharmacological evidence for such carriers has also recently been reported (33). A further interpretation of the apparently obligatory exchange process is that the carrieds) may only cross the membrane in their catecholamine-bound state, with no preference for particular a side of the membrane. Furthermore, the involvement of ATPase in an otherwise isoenergetic exchange, may indicate that the carrier(s) may be in an inactive state prior to the addition of ATP. Such activation could be due to a direct effect on the carrier or to an indirect effect via changes in ion content (e.g. protons), transport process. By contrast, the granule core per se is clearly not required for transport since granule ghosts are able to accumulate exogenous catecholamines by an ATP-dependent mechanism (9, 36-38). Nonetheless, such ghosts contain approximately 1% of their original catecholamine stores (-2 nmol/mg of protein, or more); and the question of whether transport by ghosts, at least in part, involves exchange remains to be explored. The possible existence of multiple compart- ments for transported catecholamines (e.g. aqueous phase and core) is also consistent with early observations by Slotkin et al. (39) that granules loaded with the radiolabeled catecholamines spontaneously released labeled compounds by both a fast and slow process. Considering that we have no quantita- tive understanding at present about the nature of the carrier, its interaction with ATP, or its possible functional relationship to the multiple intragranular compartments, we consider it premature to make some specific mechanistic speculations or to draw specific models. Our studies also demonstrated that at 37"C, a spontaneous efflux of epinephrine occurred from intact granules if ATP were omitted from the incubation medium (up to 3% over 2 min of incubation). In the presence of added (R)-norepinephrine, this spontaneous epinephrine efflux was in addition to that released in exchange for (R)-norepinephrine. Increasing the [MgATP] diminished epinephrine efflux, so that by.5 mm MgATP the amounts of epinephrine released and (R)- norepinephrine taken up were equal, reflecting only the exchange process (Fig. 5). The suppressive effect of MgATP did not appear to be due to reuptake of released epinephrine or to involve the granule ATPase since SITS, which inhibits the ATPase and would presumably inhibit epinephrine reuptake, had no effect on this suppression (Table 111). However, the effect of MgATP on spontaneous epinephrine efflux was prob-

6 1234 ATP-activated Catecholamine Exchange ably mediated by some granule site since it was saturable with respect to [MgATP], yielding a Km of about 4 p. This was quite distinct from the K,,, for Mg +-ATP-supported uptake (123 pl). Similar observations were made by Slotkin and Green (14) who measured catecholamine efflux from both rat and bovine chromaffin granules and found that suppression by MgATP was unrelated to reuptake or ATPase activity. They concluded that MgATP was suppressing efflux by acting at a specific membrane site. Lishajko also showed that ATP, as well as amines, diffused out of granules in the absence of added MgATP (4). It is possible that this loss of endogenous ATP may destabilize the storage complex, which results in the concomitant efflux of epinephrine. If so, then added MgATP could indirectly prevent this efflux by reducing the loss of endogenous ATP, thereby stabilizing the storage complex. Indeed, a nucleotide uptake system has been described in the chromaffin granule membrane although the K,,, for ATP was 1.4 mm (41), a value much higher than that found here for MgATP suppression of epinephrine efflux. Such a low exogenous ATP concentration could be effective at reducing ATP efflux, however, if such efflux were mediated by a membrane site having a greater affinity for ATP on the outside of the granule than on the inside. On the reasonable presumption that cytoplasmic levels of ATP are at least 1 mm, it would appear that the ATP-mediated inhibition of epinephrine efflux is probably of physiologic importance in maintaining granule stability. Another interesting question raised by this study is the basis of multiple sites of action of SITS on chromaffin granule function. SITS has previously been characterized in red cell and chromaffin granule systems as an anion transport inhibitor; yet the present work shows that it also blocks catecholamine exchange and ATPase activity. While SITS may have independent actions on these various processes, it is also possible that all of these processes might have anion require- ments. In support of this concept, we have demonstrated that chloride activates the native and detergent-solubilized granule ATPase and that both SITS pyridoxal and phosphate, another anion transport inhibitor, block the activation (42). Norepinephrine uptake is marginally (-2%) activated by 2 mm C1-, and we included C1- in our assay medium for that reason. Finally, we must consider the possible physiological value of a mechanism for ATP-dependent exchange of catecholamines in mature granules. The exchange reaction studied here might also support the replacement of endogenous (R)- norepinephrine with exogenous epinephrine, the reverse of the process studied here. This event in fact is initiated at birth in many mammals when the soluble enzyme for epinephrine synthesis, phenylethanolamine N-methyltransferase, is induced upon exposure of the adrenal medulla to newly synthesized glucocorticoids (43). The mechanism by which the induction of phenylethanolamine N-methyltransferase, a soluble enzyme, could effect quantitative conversion of granular norepinephrine to epinephrine, has remained puzzling for many years; and the reaction discussed here may provide such a mechanism. Epinephrine, synthesized in the cytoplasm by phenylethanolamine N-methyltransferase, could enter the preformed granule by exchange, freeing more endogenous (R)- norepinephrine for methylation to epinephrine without the cell having to resynthesize the entire storage organelle. This concept does not preclude other functions of the uptake system in the granule at earlier stages of assembly, in which uptake might actually result in net catecholamine accumulation. Acknowledgments-We are grateful to Bernard Lancaster for his technical assistance and to Carol Brower for preparation of the manuscript. by Chromaffin Granules REFERENCES 1. Kirshner, N. (1962) J. Biol. Chem. 237, Bashford, C. L., Casey, R. P., Radda, G. K., and Ritchie, G. A. (1976) Neuroscience 1, Johnson, R. G., and Scarpa, A. (1976) J. Gen. Physiol. 68, Winkler, H. (1977) Neuroscience 2, Pollard, H. B., Pazoles, C. J., Creutz, C. E., and Zinder,. (1979) Znt. Reu. Cytol. 58, Taugner, G., and Hasselbach, W. (1968) Naunyn-Schmiedeberg s Arch. Exp. Pathol. Pharmakol. 26, Ferris, R. M., Viveros,. H., and Kirshner, N. (197) Biochem. Pharmacol. 19, Hoffman, P. G., Zinder, O., Nikodejevik, O., and Pollard, H. B. (1975) J. Suprarnol. Struct. 4, Phillips, J. H. (1974) Biochem. J. 144, Bashford, C. L., Casey, R. P., Radda, G. K., and Ritchie, G. A. (1975) Biochem. J. 148, Casey, R. P., Njus, D., Radda, G. K., and Sehr, P. A. (1977) Biochemistry 16, Njus, D., Sehr, P. A., Radda, G. K., Ritchie, G. A,, and Seeley, P. J. (1978) Biochemistry 17, Kirshner, N. (1962) Science 135, Slotkin, T. A., and Green, H.. (1974) Biochem. Pharmacol. 23, Pollard, H. B., Zinder, O., Hoffman, P. G., and Nikodejevik,. (1976) J. Biol. Chem. 251, Pazoles, C. J., and Pollard, H. B. (1978) J. Biol. Chem. 253, Pollard, H. B., Menard, R., Brandt, H. A,, Pazoles, C. J., Creutz, C. E., and Ramu, A. (1978) Anal. Biochem. 86, Anton, A. H., and Sayre, D. F. (1962) J. Pharrnacol. Exp. Ther. 138, Itaya, K., and Vi, M. (1966) Clin. Chim. Acta 14, Jonasson, J., Rosengren, E., and Waldeck, B. (1964) Acta PhysioE. Scand. 6, Lundborg, P. (1966) Acta Physiol. Scand. 67, Carlsson, A,, Hillarp, N. A,, and Waldeck, B. (1963) Acta Physiol. Scad. 59 (Suppl. 215), Green, H... and Slotkin, T. A. (1973) Mol. Pharrnacol. 9, Viveros,. H., Arqueros, L., Connett, It. J., and Kirshner, N. (1969) Mol. Pharrnacol. 5, Da Prada. M., Obrist, R., and Pletscher, A. (1975) Br. J. Pharmacol. 53, Winkler, H. (1976) Neuroscience 1, Carlsson, A., and HiUarp, N. A. (1961) Med. Exp. 5, Johnson, R. G., Carlson, N. J., and Scarpa, A. (1978) J. Biol. Chem. 253, Cabantchik, Z. I., Knauf, P. A., and Rothstein, A. (1978) Biochim. Biophys. Acta 515, Taugner, G., and Hasselbach, W. (1966) Naunyn-Schmiedeberg s Arch. Pharrnacol. 255, Njus, D., and Radda, G. K. (1977) Biochim. Biophys. Acta 463, Johnson, R. G., and Scarpa, A. (1979) J. Biol. Chem. 254, Kanner, B. J., Fishkes, H., Maron, R., Sharon, I., and Schuldiner, S. H. (1979) FEBS Lett. 1, Njus, D., and Radda, G. K. (1979) Biochem. J. 18, Pollard, H. B., Shindo, H., Creutz, C. E., Pazoles, C. J., and Cohen, J. S. (1979) J. Biol. Chem. 254, Taugner, G. (1971) Naunyn-Schmiedeberg s Arch. Pharmacol. 27, Taugner, G. (1972) Naunyn-Schmiedeberg s Arch. Pharrnacol. 274, Schuldiner, S., Fishkes, H., and Kanner, B. I. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, Slotkin, T. A., Ferris, R. M., and Kirshner, N. (1971) Mol. Pharmacol. 7, Lishajko, F. (1969) Acta Physiol. Scand. 76, Kostron, H., Winkler, H., Peer, L. J., and Konig, P. (1977) Neuroscience 2, Pazoles, C. J., Creutz, C. E., Ramu, A., and Pollard, H. B. (198) J. Biol. Chem. 255, Wurtman, R. J., and Axelrod, J. (1966) J. Biol. Chem. 241,