THE JOURNAL OF BIOI.OGICAL CHEMLW~Y Vol. 253, No. 5, Issue of March 10, pp. 1512-1521, 1978

Printed in U.S.A.

ApH and Catecholamine Distribution in Isolated Chromaffin Granules*

(Received for publication, May 24, 1977)

ROBERT G.JOHNSON,S NANCY J. CARLSON, AND ANTONIO SCARPA§

From the Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

The effect of the proton concentration gradient (ApH) across the chromaffin granule membrane upon the rate and extent of catecholamine accumulation was studied in iso- lated bovine chromaffin granules. Freshly isolated chromaf- fin granules have an intragranular pH of 5.5 as measured by [‘%lmethylamine distribution. When stored at 4” in a well buffered medium (pH 6.90), the granules maintain this acidic intragranular pH for over 48 h, which can be ac- counted for by their large internal buffering capacity and low proton permeability. When external concentrations of 3-hydroxytyramine (dopamine) (1 to 33 mM) are added to a highly buffered suspension of chromaffin granules, there is a dose-related alkalinization of the intragranular space. Am- monium at the same concentration produces a much more rapid decrease of the transmembrane proton concentration gradient, epinephrine and norepinephrine produce a slower decrease, and 3,4-dihydroxyphenylalanine (dopa) is without effect. When the collapse of the transmembrane proton concentration gradient (ApH) is due to catecholamine addi- tion, the proton concentration gradient can be re-estab- lished by the addition of ATP. This effect is inhibited in the presence of carbonyl cyanide p-trifluoromethoxyphenylhy- drazone (FCCP), a proton translocator. Time-resolved in- flux of catecholamines into the granules was studied radio- chemically using low external catecholamine concentra- tions. Epinephrine but not 3,4-dihydroxyphenylalanine ac- cumulates under the same conditions. The rate of accumu- lation of the catecholamines depends upon the magnitude of the transmembrane proton concentration gradient. Cate- cholamine uptake is limited at pH 5.60, but the magnitude of uptake increases proportionally with increasing alkalini- zation of the external medium. Likewise, the addition of NH&l or ionophores known to collapse H+ gradients in other biological systems results in an alkalinization of the intragranular space and a corresponding decrease in the rate of accumulation of the catecholamines.

* This investigation was supported bv Grant 18708 from the National Institute of Health and Grant ?7765 from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “Wuertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of Medical Scientist Training Program Grant GM 20246.

B An Established Investigator of the American Heart Association.

It has been well established that most of the catecholamine content present in the adrenal gland is located within highly specialized granules found in the chromaffln cell (l-4). Quan- titative measurements have shown that the chromaffln gran- ules contain 1100 nmol of catecholamines/mg of protein, which corresponds to an internal concentration of 500 mM, based upon the internal water space and the assumption that all of the internal catecholamines are free in solution (5). Yet, in spite of significant research efforts in the area of catechola- mine transport, the mechanism of catecholamine accumula- tion at the molecular level is still unresolved. Two models have emerged, however, in order to stimulate further re- search.

The storage complex hypothesis states that catecholamines are capable of accumulating against an apparent concentra- tion gradient due to the fact that almost all of the catechol- amines are bound internally, uiz. with catecholamine diffu- sion occurring essentially down a concentration gradient maintained by internal binding (6-11). The active uptake hypothesis arises from Kirshner’s (12) and Carlsson’s (13) findings that net accumulation of catecholamines can occur in the presence of externally added catecholamines and ATP, presumably by means of a carrier-mediated mechanism which is directly coupled to ATP hydrolysis.

The existing experimental evidence has failed to discrimi- nate unequivocally between the two mechanisms (14, 15). In fact, it has been suggested that aspects of both models are important in the overall process of catecholamine transport (16). Thus, despite a well-documented and long-standing awareness that the chromaffin granule contains and main- tains a large accumulation of catecholamines, the molecular mechanism by which the uptake occurs remains unelicited. In this study, the effects of the magnitude of the ApH’ across the chromaffln granule membrane upon catecholamine distri- bution were investigated. In addition, the internal buffering capacity and the effect of uncouplers and reserpine upon catecholamine uptake were explored. It is concluded that catecholamine uptake may be determined wholly or at least in part by the existence and magnitude of a ApH across the chromafEn granule membrane. The results may have a deep

1 The abbreviations used are: ApH, transmembrane proton con- centration gradient: Mes. 2-(N-morpholino)ethanesulfonic acid: FCCP, carbony cyanide p-trifluoromeihoxyphenylhydrazone; dopa: mine, 3-hydroxytyramine; dopa, 3,4-dihydroxyphenylalanine; PNMT, phenylethanolamine N-methyltransferase; A+, transmembrane po- tential; Me,SO, dimethylsulfoxide; KSCN, potassium thiocyanate.

1512

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A pH and Catecholamine Distribution in Chromaffin Granules 1513

physiological relevance in terms of the homeostatic mecha- nism for cellular regulation of catecholamine stores.

EXPERIMENTAL PROCEDURES

Preparation - The chromaffin granules were isolated from bovine adrenal glands in a 0.27 M sucrose, 10 mM Tris medium, pH 7.0, and purified in a D,O/Ficoll/sucrose density gradient in order to preserve isotonicity, as described elsewhere (17, 18). The isolation medium was used for two subsequent washings and for the final suspension which was stored at 4” until use. Protein was measured by the Lowry method with bovine serum albumin as the standard (19).

Measurement of ApH- [i4C]Methylamine distribution was used to determine the ApH across the membrane of the granules as described previously (20, 21). (The concentration of methylamine added was 5.8 PM.) This technique has been applied with great success to chromaIfin granules (21), as well as other organelles such as chloroplasts (22), chromatophores (23), lysosomes (24, 25), and bacteria (26). In short, the method arises from the observation that amines freely permeate biological membranes in their neutral form (and only in this form) with equilibrium being reached when lRNH,l,inside, = [RNH,],,,,,,,,,. For amines of high pK,,

IR-NH,+l,i,,id,,I[RNH,+l OutSlde) = IH+l,inside)/[H+l(outside)

Because amines concentrate inside acidic vesicles, the ratio of the intragranular concentration to that in the medium gives a measure of the ApH. Aliquots of the reaction mixture containing [14Clmethylamine and “H,O or [i4Clpolydextran and “H,O (to correct for the extragranular water space) were centrifuged in an Eppendorf model 3200 desk centrifuge. From each centrifuge tube (a) a loo-p.1 sample was withdrawn from the supernatant, and (b) a slice was made across the bottom of the tube so that approximately half of the pellet was cut. This avoided contamination with the superna- tant. Both samples were added to 0.2 ml of 14% perchloric acid and left overnight. The acid-containing tubes were centrifuged and 0.2 ml of supernatant was added to 5 ml of Handifluor scintillation fluid (Mallinckrodt, Inc., St. Louis, MO.) containing 0.2 ml of 3 M

formic acid and counted for i4C and “H. Water content (3H,0), [14C]polydextran, and [‘4C]methylamine distribution were calcu- lated by the relative activities in the pellet and supernatant, using the equation C,,/C,,, = R + (R ~ l)[r/(l - x)], where

E = l”Clmethylamine, z = l’4C]polydextran (20 21), 3H,0 space ’ “HZ0 space

The experiments were carried out in an incubation chamber designed and constructed in the Johnson Foundation. The chamber consisted of a series of four lo-ml anaerobic cells incorporated inside a common water jacket which was connected to a Haake constant temperature circulator model. The Tri-R model MS-7 microsubmer- sible magnetic stirrer for each cell was connected to a central power supply in order that the stirring rate could be maintained constant for each cell. Anaerobic conditions were maintained by the use of nitrogen saturated solutions and a continuous flow of nitrogen through each incubation cell.

Measurement of [‘4CICatecholamine and [X’IDopa Distributions- [14C]Norepinephrine, P4C]epinephrine, [‘4C]dopamine, and [‘“Cldopa distributions were determined by the method given above for the measurement of methylamine distribution. This method produces a rapid and highly reproducible measurement of the distribution of a labeled species across a biological membrane when expressed as the ratio of the internal to external concentration. When expressed as such, the technique is limited by the unknown magnitude and extent of binding of the labeled species. In the case of methylamine, significant binding was excluded (21). In this study, the upper limit of the contribution of binding to the calculation was found to be 0.3 pH units; in most of the experiments, binding was found to be 0.1 pH units. The assumption was used that the concentration of these compounds in the external water was similar to that in the super- natant. The total concentration of each labeled compound in the incubation medium was as follows: [‘Qdopa, 23.8 PM;

[14C]norepinephrine, 12.2 p&f; [14C]dopamine, 21.9 pM; [i4C]epi- nephrine, 10.10 PM.

Measurement oflnternal Buffering Capacity- The internal buffer- ing capacity was measured by two methods. In the first, the buffering capacity of the various phases was measured directly with a standard pH electrode by the following technique: (a) the buffering capacity of 0.27 M sucrose, 2 mM Tris, adjusted to pH 5.3 with the

addition of Mes, was determined within the range pH 5.3 to 7.8 with the addition of NaOH; (b) the buffering capacity of intact granules suspended in 0.27 M sucrose, 2 mM Tris/Mes was deter- mined over the same pH range; (c) the granules were lysed by resuspension in 2 rnM Tris/Mes and the buffering capacity was measured. Experiment c gave a measure of the combined external buffering capacity of the granules, the internal buffering capacity of the granules, and the buffering capacity of the suspension me- dium. By utilizing the results of Experiments a and b, the effects of the external buffering phases could be subtracted, yielding the desired internal buffering capacity determination over the range pH 5.3 to 7.8.

In the second, various concentrations of labeled methylamine (100 pM to 50 mM) were added to chromaffin granule fractions, Since with the entrance of a neutral methylamine molecule inter- nally a protonation occurred, the number of methylamine molecules measured internally was a measure of the number of protons consumed. In addition, the entrance of sufficient methylamine molecules served to collapse the ApH. Thus, the ApH, which de- pended on the internal pH, could be determined by the ratio of the methylamine concentration inside to that outside. Since the buffer- ing capacity is measured as B, the number of H+ needed to change the pH by 1 unit/g dry weight of tissue, the measure of the number of methylamine molecules internally (i.e. the number of protons), the change in the ApH (i.e. change in the internal pH), and the dry weight of the chromaffin granules allowed for a direct computation of B. The dry weight of the granules was measured gravimetrically after drying of the chromafin granules in an oven.

Materials - Tris, Mes, epinephrine bitartrate, norepinephrine bi- tartrate, dopamine HCl, dopa HCl, reserpine, dimethylsulfoxide, and apyrase (1 unit = 1 pmol of P,/min at pH 6.5 and 30”) were purchased from Sigma Chemical Co., St. Louis, MO. FCCP was a gift of Dr. P. G. Heytler of E. I. DuPont de Nemours, Inc., Wilming- ton, Del. Nigericin was kindly supplied by Dr. J. Berger of Hoffman- La Roche, Nutley, N. J., and A23187 was from Dr. R. Hamill of Eli Lilly and Co., Indianapolis, Ind. All the ionophores were dissolved in ethanol and the volume added for each experiment did not exceed 5 pi/ml of the reaction mixture. [14C]Methylamine (10.6 mCi/mmol), [‘4C]polydextran (1.12 mCi/g), (R)-(-)-[14C]epinephrine (59.4 mCi/ mmol), (R)-(-)-[‘4C]norepinephrine (45.0 mCi/mmol), [“Cldopamine (13.69 mCi/mmol), [‘*C]dopa (12.63 mCi/mmol), and “H,O were purchased from New England Nuclear, Boston, Mass.

RESULTS

ApH and Buffering Capacity- The addition of [i4Clmethylamine to a suspension of freshly isolated chromaf- fin granules results in a large accumulation of the labeled amine (Fig. 1). Since significant binding of this amine has been previously excluded (21), the large accumulation is thought to relate to the existence of a ApH across the chromaf- fin granule membrane, inside acidic. At an external pH of 6.90, the ApH as determined by methylamine distribution is 1.4 units, indicating that the internal pH is 5.5 (Fig. 1). Ionophores and uncouplers which increase the permeability of the membrane to protons permit further investigation of the proton gradient. Since the K+ stores within the chromaffrn granule are very high (27), nigericin addition to a suspension of chromafin granules suspended in a K+-free medium cata- lyzes an exchange of an internal K+ for an external H+, and, with a sufficiently buffered external phase, an increase in the ApH is recorded due to an acidification of the intragranular space. On the other hand, when K+ is added to the external medium, the opposite exchange occurs in the presence of nigericin, uiz. K+ enters the granule in exchange for an internal H+ and the interior becomes more basic; a resultant fall in the ApH ensues. Addition of A23187, a Ca2+ ionophore known to produce a Ca2+:H+ exchange in chromaffln granules of 1:2 (21) produces a decrease in the ApH due to the exchange of inwardly directed Ca’+ with the effluxing protons. Addition of 5 mM ATP had no effect upon the ApH when added to chromaffrn granules which were freshly isolated.

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A pH and Catecholamine Distribution in Chromaffin Granules

1.5 A>OmM MgATP _--n ---

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+ 5mY K+ + Nigwicin W3pg/ml)

FIG. 1. The effect of aging on the ApH across the chromaffrn granule membrane. The reaction mixture consisted oE 0.27 M su- crose, 20 rnM Tris/Mes (pH 6.90); chromaffrn granules (10.8 mg of protein/ml); [Wlmethylamine, 3H,0; and the ionophore or ATP additions as indicated. The total incubation volume for each experi- ment was 1.2 ml. After 5 min, the samples were centrifuged in an Eppendorf desk microcentrifuge for 5 min. The supernatant and pellet were assayed for radioactivity as described under “Experimen- tal Procedures.” Temperature was 21”. The isolated granules were kept at 4” in 0.27 M sucrose, 10 mM Tris/maleate (pH 6.85) until the time of sampling. At that time, 1.2 ml of the granules were removed, washed in 0.27 M sucrose, 20 mM Tris/maleate, and resuspended in the same medium prior to incubation.

The granules were stored at 4” for several days. At varying intervals, samples were taken and the ApH measured under similar conditions. As evidenced in Fig. 1, the ApH is main- tained at a constant value over a 48-h period. Moreover, the addition of those ionophores known to perturb proton gra- dients produced similar results over the time period measured. The addition of ATP at any time over the 48-h period failed to change the ApH. Thus, the chromaffin granule appears to be able to maintain a large difference in H+ concentration across the membrane for an extended time period at low tempera- tures.

The observation of this acidic internal pH which can be maintained for an extended time at low temperatures leads to questions concerning the properties of the chromaffin granule membrane and of the internal milieu from which the proton concentration arises. It has previously been shown that the conductance of the chromafin granule membrane to protons is quite low (21). At issue is the magnitude of the buffering capacity of the internal space, that is, to determine how many protons need be added or removed from the internal space in order to change the internal concentration a given amount. The internal buffering capacity of isolated chromaffin granules expressed as the number of protons needed to change the internal pH 1 pH unit per g dry weight of granules was measured over the pH range 5.25 to 7.75 by two techniques, pH titration and methylamine distribution, as described under “Experimental Procedures” (Fig. 2). The internal buffering capacity is biphasic from pH 5.25 to 7.00, corresponding to a higher buffering capacity at low pH and a significantly lower buffering capacity at a higher pH.

At pH 5.5, corresponding to the internal pH at the time of granule isolation, the buffering capacity approaches 300 pmol of H+/pH unit/g dry weight of granules; while at an intragran- ular pH of 6.25, there is a 3-fold difference, with the buffering

Ob 5.50 6.00 6.50 7.00 7.50 a00

FIG. 2. The internal buffering capacity of isolated chromaffrn granules expressed as micromoles of H+/pH unit/g dry weight granules. The buffering capacity was determined by two methods: titration (O-O) and methylamine distribution (W-m) as de- scribed under “Experimental Procedures.” Temperature was 24”.

Minutes

FIG. 3. Time course of the ApH changes after addition of various concentrations of dopamine. The reaction mixture contained, in addition to the dopamine concentration indicated; 0.27 M sucrose, 40 mM Tris/maleate (pH 6.85); chromaffrn granules (14 mg of protein/ ml); [Wlmethylamine; and 3H,0. The total initial volume was 10 ml. At the times indicated 1.2-ml samples were taken and centri- fuged in an Eppendorf desk microcentrifuge for 5 min. The superna- tant and pellet were assayed for radioactivity as described under “Experimental Procedures.” ATP, 10 mM, was added after 2 h, as indicated. Temperature was 23”.

capacity approaching 100 pmol of H+/pH unit/g dry weight. Catecholamine Influx and ApH- When 33 mM dopamine is

added to a suspension of chromaffm granules, there is a time- dependent decrease in the ApH (Fig. 3). The rate and extent of the decrease in the ApH is related to the concentration of dopamine added. Since the external phase is well buffered, the fall in the ApH represents an alkalinization of the intra- granular phase. The addition of 33 mM dopamine causes a rapid fall in the ApH, while the addition of 105 PM or 1 mM dopamine results in a minimal fall. ATP addition, after 60 min of incubation, results in a rapid reestablishment of the ApH, in certain cases approaching the original intragranular pH value. Dopa, the precursor of dopamine in the biosynthetic pathway, does not collapse the ApH when added at the same concentration (Fig. 4). Dopa, like dopamine, norepinephrine, and epinephrine, contains an amine group. However, unlike these three catecholsmines, dopa also possesses a carboxylic acid group. Norepinephrine and epinephrine, the P-hydroxyl- ated epimers of dopamine, also cause a decrease in the ApH when added at the same concentration. However, the rate of

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ApH and Catecholamine Distribution in Chromaffin Granules 1515

the decrease is significantly less than that of dopamine. Again, ATP addition causes a reestablishment of the ApH.

When 5 mM ATP is added at the start of the incubation in the presence of dopamine (33 mM) the fall of the ApH is inhibited (Fig. 5). In fact, the collapse of the ApH can be retarded for varying amounts of time, depending upon the concentration of ATP added (data not shown).

FCCP is a compound which transports H+ electrogenically across artificial and biological membranes. Although the addition of FCCP has been shown to be capable of increasing the permeability of a membrane to protons, in the absence of a permeable anion or cation the movement of a charged proton establishes a diffusion potential which limits the fur- ther efflux of protons. When added to a suspension of chromaf- fin granules including 33 mM dopamine, FCCP only slightly increases the rate of collapse of the ApH (data not shown).

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FIG. 4. The effect of various amines upon the ApH in isolated chromaffin granules. Chromafin granules were added to a reaction mixture of 0.27 M sucrose, 45 rn~ Tris/maleate (pH 6.80); 33 rnM dopa, norepinephrine, epinephrine, or dopamine; [Wlmethylamine; and 3H,0. No amines were added in the control experiment. The isolation and assay procedures were similar to that of Fig. 1. Temperature was 24”.

ADH II r-

5mM ATP

Time him&s)

FIG. 5. Time-dependent changes in the ApH in the presence of (a) no additions (O-O), (5) 33 mM dopamine (A-A), and (c) 33 rnM dopamine + 5 rnM ATP (m- - -m). The reaction mixture con- sisted of chromaffin granules (13.3 mg of protein/ml), 0.27 M sucrose, 45 rnM Tris/maleate (pH 6.88, [Wlmethylamine, 3H,0, and the additions as indicated. Total initial volume was 10 ml. Samples, 1.2 ml, were centrifuged and assayed as given in Fig. 3. At 90 min, as shown by the arrows, 5 mM MgATP was added to the various reaction mixtures, and in one experiment, FCCP (3 p/ml) was added. Temperature was 24”.

However, in the presence of FCCP at the time of ATP addition after 90 min (Fig. 5), the reestablishment of the ApH is abolished and the ApH remains at a low value.

NH&l, (NHJ2S04, or methylamine addition at a concentra- tion of 33 mM produces a rapid fall in the ApH which reaches a steady state level in less than 5 min. The collapse in the presence of these amines is not sensitive to the addition of ATP at 60 min (data not shown).

External pH and Catecholamine Distribution - In order to resolve the time course of the influx of catecholamines into the chromaffin granules, experiments were undertaken using labeled catecholamines of very limited concentration. The use of concentrations of catecholamines which do not perturb the ApH allows direct determination of catecholamine uptake and ApH under circumstances where the H+ gradient is independently enhanced or collapsed.

The time-dependent accumulation of epinephrine as mea- sured by the distribution of the i4C label is shown in Fig. 6. This accumulation is kinetically resolvable, differing from that of methylamine distribution (used to measure the ApH), which is virtually instantaneous. On the other hand, the figure illustrates that dopa, the dopamine precursor and an acid, does not accumulate.

Since the internal pH of the chromaffin granules is main- tained at pH 5.5, suspending the chromaffin granules in a medium of the same pH should result in a very small H+ gradient across the membrane. As can be seen (Fig. 7), when the external pH is 5.60, the ApH measured by methylamine distribution approaches 0.1. At this pH, the accumulation of dopamine and norepinephrine is limited. When NaOH is added such that the external pH is raised from pH 5.60 to pH 6.20, as measured with a pH electrode, there is a rapid re- equilibration of methylamine, corresponding to a change in the ApH from 0.1 to 0.6 pH units. Commensurate with the alkalinization of the external phase is a rapid but kinetically resolvable uptake of dopamine and norepinephrine into the chromafin granules.

Increasing the pH toward the pK, of the amines, however,

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FIG. 6. Time-resolved accumulation of [Wlepinephrine, [Wldopa, and [Wlmethylamine expressed as the logarithm of the ratio of the internal to external concentrations. In addition to chromaflin gran- ules (10.5 mg of protein/ml), the incubation mixture contained: 0.27 M sucrose, 15 mM Tris/maleate (pH 6.95); W-labeled amine; and 3H,0. At the times indicated, 1.2-ml samples were taken and the procedure outlined in Fig. 3 was followed. Temperature was 36”.

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1516 A pH and Catecholamine Distribution in Chromaffin Granules

increases the population of the uncharged and presumably permeable form. Therefore, uptake was also measured at various external pH values under conditions where the con- centration of uncharged amine was held constant (Fig. 8). Over the time period studied, the rate of epinephrine accu- mulation is determined by the external pH with a rapid uptake occurring at pH 6.71 and limited uptake at pH 5.55 (Fig. 8A). The rate of accumulation of epinephrine, dependent upon the external pH even when the uncharged species is maintained at the same concentration at each pH value, is also reflected in the ApH, which is large at pH 6.71 and minimal at pH 5.60 (Fig. 8B).

When dopamine accumulation is measured after 60 min, at which time the accumulation approaches a steady state, the distribution of dopamine is dependent upon the external pH in a linear fashion under conditions wherein either equal concentrations of the uncharged form of dopamine are present at each pH (Fig. 9A), or the total concentration of charged plus uncharged form is equal at each pH (Fig. 9B). In addition, the dopamine accumulations at any given pH for

FIG. 7. Time course of [14C]methylamine, l’4Cldopamine, and [14Clnorepinephrine distribution after pulsed base titration. The reaction mixtures contained: 0.27 M sucrose, 10 mM TrislMes (pH 5.60); chromaffin granules (20.1 mg of protein/ml); lL4CJmethylamine, 114Cldopamine, or [‘4Clnorepinephrine; and 3H,0. At 18 min (arrow) NaOH was added so that the external pH increased from pH 5.60 to pH 6.20 as measured with a pH electrode. The reaction mixture aliquots were treated as described in Fig. 3. The methylamine distribution expressed as log C,,/C,,, is taken as a measure of the ApH. In the absence of NaOH addition, the norepinephrine distri- bution ratio after 68 min was 0.26. Temperature was 34”.

Tlmo (mhl**, mm. bcdJ!um)

FIG. 8. Distribution of [14Clepinephrine as a function of the ApH. The reaction mixtures contained: 0.27 M sucrose, 30 mM Tris/Mes at the indicated pH; chromaffin granules (10.9 mg of protein/ml); 3H20; and either [14C]epinephrine (A) or [‘*C]methylamine (B). In A, the epinephrine concentration was adjusted so that at each pH the uncharged form was present at the same concentration, using a pK, value for the ammonium group of 10.5. The added concentrations of epinephrine were as follows: pH 5.5, 404 PM; pH 6.21, 78 PM; pH 6.71, 25.1 PM. The experiment performed was similar to that of Fig. 3. Temperature was 36”.

both conditions, equal concentrations of uncharged dopamine or equal concentrations of total amines, are approximately equal.

Internal pH and Catecholamine Distribution - It has previ- ously been shown that the internal pH of the chromaflin granules is increased after the addition of A23187 and Ca’+ (Fig. 1). In this experiment (Fig. lo), the ApH was decreased to 1.2 pH units (and thus the internal pH rose to pH 6.0) upon the addition of A23187 and Ca*+ (Fig. lOA). Raising the internal pH 0.5 pH units (and thus decreasing the ApH) dramatically reduced the rate of dopamine accumulation (Fig. 1OB).

The effect of the internal pH upon catecholamine distribu- tion was also determined by increasing the internal pH 20 min after the onset of [‘%lepinephrine accumulation. At this point (Fig. 11) the ApH was collapsed by NH&l, K+ plus nigericin, or A23187 plus Ca*+. With all of the additions, the rate of epinephrine accumulation was dramatically decreased (Fig. 1IA). The effect of the addition upon the ApH is also shown (Fig. 1IB).

The effect of collapsing the ApH after reaching a steady state accumulation of catecholamines was also investigated (Fig. 12). The addition of NH&l produces a rapid and large redistribution of methylamine which is consistent with the measurement of a small ApH. However, the collapse of the ApH does not result in a redistribution of epinephrine. Simi- larly, the addition of A23187 and Ca2+ results in a large efllux of methylamine and corresponding decrease in the ApH (Fig. 13). But the decrease in the ApH has only a minimal effect upon the dopamine and norepinephrine distributions.

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FIG. 9. Distribution of 114C1dopamine as a function of the ApH under conditions where (a) the concentration of the uncharged species was the same at each pH (A), and (b) the total concentration of dopamine (charged plus uncharged species) was the same at each pH. In addition to chromaffin granules (10.1 mg of protein/ml), the reaction mixture contained: 0.27 M sucrose, 30 rnM Tris/Mes adjusted to the indicated pH; l’4Cldopamine; and 3H,0. The incubation volume of each sample was 1.2 ml. The pK, of the ammonium group was taken as 10.5. The added concentrations of dopamine in A were as follows: pH 5.5, 446 PM; pH 6.0, 141 PM; pH 6.5, 44.6 PM; pH 7.0, 14.1 pM; and pH 7.5, 4.46 PM. In B, the added concentration was 4.46 PM. After 60 min, the samples were centrifuged for 5 min in an Eppendorf desk microcentrifuge, after which the pellet and super- natant were assayed for radioactivity as described under “Experi- mental Procedures.” Temperature was 24”.

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ApH and Catecholamine Distribution in Chromaffin Granules 1517

- 0 60

Time (minks)

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FIG. 12. The effect of decreasing the ApH upon steady state [Wlepinephrine and [Wlmethylamine distribution. The reactibn mixture consisted oE chromaffrn granules (13.8 mg of protein/ml); 0.27 M sucrose, 30 rnM Trislmaleate (pH 6.85); 3H,0; and either [Wlmethylamine or [Wlepinephrine. At 68 min (arrow) 33 mM NH&l was added to each reaction mixture. Total initial volume was 10 ml. Samples, 1.2 ml, were centrifuged and assayed as described in Fig. 9. Temperature was 36”.

Reserpine and Catecholamine Accumulation - Reserpine has been utilized as a pharmacological agent in the study of catecholamine uptake and release in viva and in vitro. There have been conflicting reports as to whether reserpine inhibits the binding and translocation of catecholamines (28, 30) or ATP hydrolysis in intact granules (31,321. In the experimental

1.4 r

FIG. 10. The effect of alkalinization of the intragranular space upon [‘Cldopamine accumulation. The re- action mixtures contained: chromaffrn granules (23 mg of protein/ml); 0.27 M sucrose, 30 mM Tris/Mes (pH 7.2); 3H,0; [WJmethylamine (A) or [Wldopamine (21.9 PM) (B); and cal- cium plus A23187 as indicated. Sepa- ration and assay procedures were sim- ilar to those described in Fig. 9. Tem- perature was 35”.

FIG. 11. Kinetics of epinephrine ac- cumulation as a function of the inter- nal pH. Chromaffrn granules (10.8 mg of protein/ml) were added to a suspen- sion consisting of: 0.27 M sucrose, 30 mM Tris/maleate (pH 6.9); 3H20; and either [“Clepinephrine (4 or [Wlmethylamine (B). After 18 min, the internal pH was increased by the addition of 33 rnM NH&l, 15 mM K+ plus nigericin (3.1 pg/ml), or 400 pM CaCl, plus A23187 (18 pg/ml). Samples were treated as described in Fig. 9. Temperature was 35”.

0 IO 30 30 m so Time (minutes 1

FIG. 13. The effect of increasing the internal pH upon [Wlnorepinephrine, [Wldopamine, and [Wlmethylamine distri- butions. The reaction mixture contained, in addition to chromaffrn granules (10.4 mg of protein/ml): 0.27 M sucrose, 30 mM Tris/maleate (pH 6.85); 3HZO; and the indicated W-labeled amine. After 64 min (arrow), 400 pM CaCl, plus A23187 (6.6 pg/ml) were added to each sample. Centrifugation and assay for radioactivity were similar to that described in Fig. 9. Temperature was 36”.

system utilized, reserpine showed a dose related inhibition upon catecholamine uptake (Fig. 14). At the concentrations of reserpine added, there was no effect upon the ApH (data not shown).

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1518 A pH and Catecholamine Distribution in Chromaffin Granules

/

Control

// Plus Reserpine LOS&ml)

Plus Rcserpine (0.6pg/mt)

Plus Reserpine (6~glml)

OOWO Minutes

FIG. 14. The effect of reserpine on epinephrine accumulation in isolated chromaffin granules. Chromaffm granules (11.8 mg of protein/ml) were added to a suspension medium of: 0.27 M sucrose, 30 rnM Tris/maleate (pH 6.85); 3Hp0; [“Clepinephrine, and the indicated concentration of reserpine. The reserpine was dissolved in MeTSO. The addition of reserpine to the reaction mixtures consti- tuted less than l/40 of the total volume. Me%SO alone had no effect upon epinephrine accumulation. Samples were centrifuged and assayed as described in Fig. 9. Temperature was 35”.

DISCUSSION

ApH and Buffering Capacity - The ApH which is measured across the chromaffin granule membrane immediately after isolation of the granules retains a constant magnitude over 48 h (Fig. 1) if the granules are kept at 4”. Since it is very unlikely that the internal H+ concentration is being replen- ished by an energetic process at this temperature, the main- tenance of a longstanding proton gradient reflects the very low conductance of the membrane to protons at this tempera- ture as well as the high buffering capacity at an internal pH of 5.5. In a previous report (21), it has been shown that the conductance of the chromaffin granule membrane to protons may be lowest of any previously isolated subcellular organelle. In this report, it has been shown that a substantial internal buffering capacity exists in the range where the intracellular pH achieves its lowest value (Fig. 2). Thus, the long term maintenance of the ApH may well be derived from these two complementary factors, i.e. few protons can efflux from the granules because the permeability of the granules to protons is quite low, and those protons which are removed from the intracellular space do not greatly influence the internal proton concentration because the buffering capacity is very high.

The values obtained for the internal buffering capacity (Fig. 2) compare with values from other organelles as follows: for mitochondria 88 pmol H+/pH unit/g dry weight (331, and for Micrococcus denitrificans 90 wmol of H+/pH unit/g dry weight (34). The extremely high buffering capacity of the chromaflin granules at pH 5.5 may partially reflect itself in the observation that ATP addition to recently isolated gran- ules suspended in a medium at pH 6.95 produces no change in the internal pH (Figs. 1 and 5). This observation probably does not relate to a limitation in the free energy of hydrolyses of ATP in translocating protons, since a similar negative effect is seen when the external pH is 5.5 (data not shown). Physiologically, the presence of a large buffering capacity in the region where the internal pH is maintained would tend to protect the internal pH from rapid pH changes in the face of a large influx of amines, which would tend to consume H+ and decrease the ApH.

Catecholamine Influx and ApH-It has been previously

proposed that the physiological significance of a large proton gradient across the chromaffin granule membrane may be 3- fold (21). One suggestion was that the acidic intragranular space and resultant ApH may exist as the basis of the fundamental mechanism of catecholamine accumulation. Be- cause catecholamines are primary or secondary amines with a high pK,, and if catecholamines can accumulate via the same process as methylamine, i.e. permeation in the neutral form, then it follows that catecholamines should also be able to accumulate against a concentration gradient as determined by the ApH.

The rapid fall in the ApH upon addition of dopamine (Fig. 3) provides a striking illustration that catecholamine permea- tion of the chromaffin granule membrane can occur in the uncharged form. Moreover, the time dependence of the col- lapse reflects the intrinsic permeability of the chromaffin granule membrane to dopamine (the collapse of the ApH in the presence of NH&l is almost instantaneous).

The slower permeation of epinephrine and norepinephrine at the same concentration as that of dopamine reflects the lower intrinsic permeability of the chromaffin granule mem- brane to these species (Fig. 4). Since dopamine differs from norepinephrine and epinephrine by the presence of a P-hy- droxyl group, the presence of this group may greatly retard catecholamine permeation. Similar effects, i.e. slower permea- tion of epinephrine and norepinephrine versus dopamine, have been noted in the permeation of these compounds into liposomes across the membrane of which an artificial pH gradient was induced (35). The concentration dependence of dopamine upon the ApH collapse (Fig. 3) reflects the internal buffering capacity. Significantly, dopamine concentrations of up to 1 mM have no effect upon the ApH. The addition of ATP after 60 min (Figs. 3 and 4) produces a re-establishment of the ApH. This ATP addition is associated with a rapid ATP hydrolysis.

These results suggest that ATP hydrolysis provides the energy necessary for H+ translocation, the newly appearing H+ in essence serving to replace the internal H+ which was bound by the internally accumulated catecholamine. How- ever, in the presence of FCCP, which makes the membrane freely permeable to protons and establishes a diffusion poten- tial in a direction presumably opposite to that of the inwardly directed H+ translocation mediated by the ATPase, no reaci- dification occurs.

The addition of oligomycin, an inhibitor of the mitochon- drial ATPase, had, unlike FCCP, no effect upon the reacidifi- cation when added concurrent with ATP.

External pH and Catecholamine Distribution- When the accumulation of catecholamines is measured directly using labeled catecholamines (Fig. 6), qualitatively similar results are obtained as those inferred from the previous experiments (Figs. 3 to 5). There is a time-dependent accumulation of epinephrine such that after 40 min the internal concentration of epinephrine is 10 times that of the external concentration. On the other hand, dopa is not accumulated. While specula- tive, these results may have important physiological implica- tions. The existence of an acidic intragranular space serves as a mechanism by which basic compounds ran accumulate. Acidic compounds, however, would tend to be excluded. Both the precursors of dopamine, dopa (36-38), and the predomi- nant metabolites of the catecholamines 3,4-dihydroxyphen- ylacetic acid and 3,4-dihydroxymandelic acid (39, 40) are acidic compounds and would tend to be excluded from the intragranular space. Thus, the existence of the ApH may

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A pH and Catecholamine Distribution in Chromaffin Granules 1519

allow for the accumulation of only those compounds which are capable of biosynthesis to biologically active compounds.

A number of experiments were presented in which the external pH was varied (Figs. 7 to 9). When the external pH approached the internal pH (Fig. 7) there was a minimal accumulation of dopamine and norepinephrine. Only when the external pH was raised over 0.5 pH units did an influx of these two amines persist. This influx of catecholamines was shown not to be dependent upon the population of uncharged amines (Fig. 81, rather to be dependent upon the magnitude of the ApH (Fig. 9).

Reserpine addition resulted in a dose-dependent inhibition of catecholamine accumulation (Fig. 14). Over the last decade, thousands of papers have been reported dealing with the mechanism of action of reserpine (for review see Ref. 41). In spite of the magnitude of the effort, the mechanism of reser- pine action remains undefined. The existing evidence suggests that reserpine may inhibit catecholamine accumulation by binding to a catecholamine membrane carrier. On the other hand, it has been shown that reserpine can affect the transport properties of other noncatecholamine accumulating organelles such as Ca*+ transport by sarcoplasmic reticulum vesicles, presumably by perturbation of the lipid moiety (42). Both possibilities are consistent with the experimental evidence, and further research efforts using reserpine may allow deter- mination of the molecular mechanism of catecholamine trans- location at the membrane level.

Znternal pH and Catecholamine Distribution - Raising the internal pH resulted in a decreased rate of uptake of catechol- amines (Fig. 10). Likewise, when the internal pH was raised after 20 min of incubation by NH&l, nigericin plus K+, or A23187 plus Ca*+, a striking decrease in the rate of epineph- rine uptake resulted (Fig. 11). Since raising the internal pH is tantamount to a decrease in the ApH, these experiments also suggest catecholamine accumulation is sensitive to the ApH.

Alkalinization of the intragranular space after steady state accumulation of epinephrine, norepinephrine, or dopamine was reached failed to produce any change in the distribution of these amines (Figs. 12 and 13). This is opposed to the effect of intragranular alkalinization on methylamine which ef- fluxes from the chromaffin granule in the face of a decrease in the ApH, and re-equilibrates at the new ApH value. These results support the well established observation that catechol- amines which accumulate inside chromaffin granules are tightly bound (43-46). Thus, it is clear that catecholamines do not at steady state distribute according to the ApH, as does methylamine, due to the very strong internal binding capacity for catecholamines which does not exist for smaller amines. However, it is strongly suggested by the experiments pre- sented here that catecholamine accumulation is intimately involved with the magnitude of the ApH across the chromafin granule membrane.

ATP, ApH, and Catecholamine Distribution - When ATP is added to a suspension of chromaffin granules suspended in a medium containing a labeled catecholamine, the rate of uptake of the catecholamine is enhanced at least 8- to lo-fold over the rate of accumulation in the absence of ATP (data not shown). However, results presented here (Fig. 1) demonstrate that ATP addition has no effect upon the ApH in freshly isolated chromaffin granules.

Preliminary experiments using S’CN distribution (data not shown) show in accordance with previous reports that the addition of ATP results in the establishment of a membrane

potential, inside positive, across the chromaffin granule mem- brane (47). Collapse of the membrane potential with FCCP or KSCN addition results in inhibition of catecholamine accu- mulation even as ATP hydrolysis persists. In addition, the increased rate of uptake is also dependent upon the magnitude of the ApH, since collapse of the ApH by NH,Cl or A23187 and Ca*+ dramatically inhibited the accumulation of the labeled catecholamines in the presence of ATP. These early results suggest that catecholamine uptake in the presence of ATP is directly dependent both upon the existence of a ApH and a A+. One possible explanation is that the catecholamines must obligatorily permeate the membrane in the neutral form as occurs in the absence of ATP, their permeation thus being dependent upon the ApH. Another component, perhaps a carrier molecule, may be potential-dependent. Such a compo- nent would explain the rapid increase in accumulation of catecholamines when ATP is added. Ultimately it may be shown that catecholamine accumulation via an ATP-mediated process occurs entirely unrelated to permeation of catechol- amines as a neutral species. However, this study has shown that accumulation as the neutral species does occur and even as an ATP-independent process may play a significant role in the overall mechanism of net uptake of catecholamines. Sub- sequent work will delineate the precise role which the ApH has upon catecholamine accumulation, i.e. whether accumu- lation obligatorily occurs in the uncharged form even in the presence of ATP, or whether accumulation by the uncharged form occurs via a separate mechanism distinct from the uptake resulting when ATP is added, and which presumably is potential-dependent.

The effect of any endogenous ATP released from damaged or leaky granules upon catecholamine accumulation is thought to be minimal, based upon the following control studies (data not shown). First, the addition of apyrase (5.0 units/ml of incubation medium) did not affect the rate or extent of catecholamine accumulation. Second, FCCP addition (1 pg/ml), which was shown to inhibit the effect of ATP on reacidification of the internal space (Fig. 51, had no effect upon the distribution of [‘%!lepinephrine, [‘4Clnorepinephrine, or [14Cldopamine.

The experimental evidence also suggests that the distribu- tion of labeled catecholamines is not due to exchange. First, in all of the experimental conditions, (a) increasing or decreas- ing the external pH (Figs. 7 and 81, (b) increasing the internal pH (Figs. 10 and ll), or (c) varying the external catecholamine concentration (Fig. 91, the rate of catecholamine accumulation was directly related to the ApH. Second, it has been shown that increasing the internal pH results in an efflux of cate- cholamines (21). However, the rate of accumulation of labeled catecholamines decreased under this condition (Figs. 10 and 11). Third, it is well known that catecholamine efflux in- creases considerably at lower pH values (48, 49). However, at pH 5.5, the rate of catecholamine accumulation was found to be minimal (Fig. 7).

Models for Catecholamine Uptake - The two models found in the literature are presented schematically in Fig. 15. The fundamental postulate of the storage-complex hypothesis is that ATP and catecholamines form a strong intragranular storage complex based upon electrostatic interactions of nega- tively charged ATP with positively charged catecholamines. Catecholamine uptake occurs down a concentration gradient maintained by the internal binding. In the active uptake hypothesis, catecholamine uptake is thought to occur by a carrier mediated process directly linked to ATP hydrolysis

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1520 A pH and Catecholamke Distribution in Chromaffin Granules

FIG. 15. Models of catecholamine uptake into isolated chromaffrn granules.

and occurs against a concentration gradient. Utilizing the results of this study and others, it is possible

to propose a third model, the H+ ion gradient hypothesis. The essential feature of this model is that catecholamine uptake occurs as a result of the existence of a proton gradient and the basic property of catecholamines. Thus, catecholamine influx occurs with the catecholamine in the neutral form. Once inside the neutral species becomes reprotonated. Since the influx of a sufficient quantity of catecholamines would tend to consume H+ and raise the internal pH, a mechanism must exist by which the proton gradient is maintained. Conceptually, this may manifest as the translocation of an H+ across the chromaffin granule membrane by a process dependent upon utilization of ATP. Evidence has been pre- sented in this study that catecholamine uptake may be de- pendent upon the magnitude of the ApH. In addition, indirect evidence has been shown here and elsewhere (50, 51) that the event concurrent with ATP hydrolysis may be proton translo- cation. The proton translocating system would allow for the generation of an acidic intragranular space and preserve the internal pH in the face of the catecholamine accumulation which would tend to collapse it.

The model as presented, although simplistic, nonetheless is able to explain previous experimental results which were particularly difficult to reconcile with other models. First, it has been well substantiated that there is no fixed stoichiome- try between catecholamine uptake and ATP hydrolysis (52, 53). By the H+ ion model, no stoichiometry would be predicted since ATP hydrolysis and catecholamine transport are not directly coupled processes. Second, it has been reported that the pH optimum for transport of catecholamines is different from the Mg’+ATPase activity of the granule membrane (54). In fact, at high pH values, the ATPase activity decreases while the rate of transport increases. The model predicts that as the ApH increases, the rate of catecholamine accumulation should likewise increase. The pH optimum for the ATPase activity reflects the endogenous properties of the ATPase,

and, if sufficient to maintain the ApH, would not limit the rate of catecholamine accumulation. Third, reserpine addition inhibits binding or translocation of catecholamines, or both, and has no effect upon ATP hydrolysis (28, 31). Again, since catecholamine accumulation and ATP hydrolysis are discrete processes, the action of reserpine is unilateral.

The existence of an ATP driven proton pump has been well documented in a wide variety of cellular and subcellular organelles (55). The resultant transmembrane pH gradient and membrane potential, collectively termed the protonmotive force, have been implicated in many energy requiring func- tions of these biological membranes. Conceptually, the in- wardly directed H+ translocation for chromaffin granules is similar to that found for chloroplasts which also are capable of generating and maintaining an acidic interior.

In the context of the results presented, no carrier for catecholamine translocation need be postulated. It has been shown using liposomes that catecholamines accumulate intra- vesicularly if the internal space is acidic (35). Since by definition no protein moiety exists within the liposome, per- meation of the lipid milieu, presumably by the uncharged catecholamine molecule, must suffice. Translocation of cate- cholamines across the chromaffin granule membrane, based upon the intrinsic permeability of the lipid phase, may allow net accumulation of catecholamines at extremely low cytosolic levels, since distribution would be limited only by the magni- tude of the ApH.

Acknowledgment -Many thanks to Ms. Ann Hickey for typing the manuscript.

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