371

J. Phyeiol. (1977), 269, pp. 371-394 With 9 text-ftguree Printed in Great Britain

CALCIUM MOVEMENTS DURING THE RELEASE OF CATECHOLAMINES FROM THE ADRENAL MEDULLA: EFFECTS OF METHOXYVERAPAMIL AND EXTERNAL CATIONS

BY J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFARO From the Department of Pharmacology and Therapeutic, McGill University, Montreal H3G 1 Y6, Quebec, Canada

(Received 4 November 1976) SUMMARY

1. Cortex-free adrenal glands previously labelled with the isotope "Ca have been perfused with Locke or modified Locke solution to assess Ca2+ movements under different conditions. 2. Substitution of Na+ by either sucrose or choline during perfusion with Ca2+-free Locke solution induced a significant and sustained decrease in the 4"Ca effilux. Concomitant with this effect there was an increase in the output of catecholamines from the perfused gland. 3. In the presence of Ca2+ (2.2 mM) in the perfusion fluid, Na+ omission induced an increase in the 45Ca efflux. This increase was significantly reduced if 3 x 10-4 M methoxyverapamil (D-600) was present in the perfusion fluid. However, the increased catecholamine output in response to Na+ deprivation remained unchanged. 4. Excess of Mg2+ (20 mM) in the extracellular medium blocked the increase in catecholamine output in response to Na+ omission. However, the decrease in the 4"Ca efflux produced by Na+ deprivation in the presence of this high concentration of Mg2+ was similar to that observed in the presence of 1-2 mM-Mg2+. 5. In the absence of Mg2+ in the extracellular medium, substitution of Na+ by either sucrose or choline induced a sharp and transient increase in the 45Ca efflux rate coefficient. This increased 4"Ca efflux, which has similar time course as the enhanced catecholamine output, was not affected by the presence of 3 x 10-4 M methoxyverapamil. 6. In the absence of Mg2+, the graded substitution of Na+ in the perfusion medium by sucrose enhanced the efflux of "Ca. This increase in the "Ca outward movement was linearly related to the logarithm of the extracellular Na+ concentration. 7. After perfusion of glands with Ca2+-free Locke solution, the

372 J. AGUIRRE, J. E. B. PINTO AND J. M. TRJFAR6 reintroduction of Ca2+ (2.2 mM) into the perfusion fluid produced an increase in the "Ca efflux. This was accompanied by a discharge of catecholamines. 8. Although Mg2+ (20 mM) was effective in blocking catecholamine release, this divalent cation did not modify the increase in the "5Ca efflux produced by Ca2+ reintroduction. 9. In contrast to these later observations, methoxyverapamil (3 x 10-4 M) was effective in inhibiting both increases in catecholamine output and 45Ca efflux in response to Ca2+ reintroduction. 10. It is concluded from these experiments that (a) Ca2+ movements in the adrenal medulla may involve both Na+ - Ca2+ and Ca2+ -Ca2+ exchange mechanisms; (b) the omission of Na+ from the extracellular environment produces not only an increase in the output of catecholamines but it may increase the intracellular levels of Ca2+ and that this may result in an increased Ca2+ efflux when Mg2+ is omitted from the perfusion fluids, and that (c) the competition between Ca2+ and Mg2+ during the secretary process may involve an intracellular site. INTRODUCTION

The role of extracellular Ca2+ in stimulus-secretion coupling is well documented (Douglas, 1968; Smith & Winkler, 1972). Transmitter release can be induced by intracellular administration of Ca2+ (Miledi, 1973) and this suggests that the intracellular concentration of free Ca2+ is one of the main regulatory factors involved in the secretary process. It has been shown that, unlike during acetylcholine stimulation, Na+ deprivation induces exocytotic release of catecholamines from the adrenal medulla in the absence of extracellular Ca2+ (Lastowecka & Trifar6, 1974). Therefore, unless different types of stimulation trigger exocytosis by different cellular and molecular mechanisms, Na+ omission must induce an increase in intracellular Ca2+. To test this possibility and in order to understand further the role of Ca2+ and Na+ in the release process, a study on the movement of Ca2+ in the adrenal medulla was carried out. The results reported in this paper on the efflux of Ca2+ from adrenal glands during catecholamine release induced by Na+ deprivation, support the idea that intracellular Ca2+ is involved in the release reaction. A preliminary account of some of these results has been given elsewhere (Pinto, Trifaro6 & Cardinal, 1975). METHODS

Bovine adrenal glands obtained from a slaughterhouse were freed from their cortices and perfused in vitro with Locke solution as described by Trifar6, Poisner & Douglas (1967). The composition of the perfusion fluids was (mM): (a) regular Locke solution, NaCl, 154; CaCl2, 2-2; MgCl2, 12; KC1, 2.6; K2HPO4, 2-15; KH2PO4, 0-85; dextrose, 10; (b) Na+-deficient and Na+-free Locke solutions were of the same

CALCIUM EFFLUX IN THE ADRENAL MEDULLA

373

composition as the standard Locke solution except that NaCl was partially or totally replaced by equimolar concentrations of LiCl, choline chloride or osmotically equivalent concentrations of sucrose; (c) Ca2+-free Locke and Na+-free, CaS+-free Locke solutions were similar to the solutions mentioned in (a) and (b) respectively, except that CaCl, was omitted from the media. In the experiments in which methoxyverapamil (D-600) was tested the perfusion fluids contained 0-06 % ethanol which did not affect either the spontaneous or the evoked 4"Ca efflux. All solutions were equilibrated with 5 % CO2 in 2 and the final pH of the solutions was 7 2. The glands were perfused at room temperature (250 C) using a multichannel peristaltic pump (Bfichler) at a constant rate (10 ml./min). Each gland was perfused for 20 min with normal Locke solution (equilibration period) and, in order to wash out the extracellular Ca2+, perfusion was switched to Ca2+-free (EDTA, 2 mM) Locke for another period of 25 min. Perfusion was then continued for 5 min with the same Ca2+-free solution but devoid of EDTA. This was followed by a 4 min period (labelling phase) in which the glands were exposed to 45Ca, 5 ,Uclml. (specific activity: 2-27 pscln-mole). After this labelling phase, perfusion was continued with Ca2+-free Locke medium for 50-80 min to washout extracellular 4"Ca. In some experiments the time required to wash out the extracellular space was determined after exposing the glands for 4 min to [14C]sorbitol, 1 pc/ml. (specific activity: 200 pcfcpmole). After the radioactive pulse samples of the perfusates were collected for a period of 1 min at 1 or 2 min intervals by a fraction collector (Bchler). Aliquots (0.5 ml.) of each sample were transferred to vials containing 15 ml. Aquasol (New England Nuclear, Boston, Mass.), and the radioactivity was measured in a liquid scintillation spectrometer (SL 40 Intertechnique). The "Ca remaining in the tissue at the end of each experiment was determined after ashing pieces from different portions (i.e. middle and both ends) of the gland. These pieces were weighed, blotted on filter paper, and transferred into silica crucibles which were then placed in a muffler furnace (6000 C) for 18-24 hr. The ashes were resuspended in concentrated nitric acid and the radioactivity determined as indicated above. Desaturation curves were plotted from the 46Ca values determined in the effluents after adding the radioactivity remaining in the gland at the end of the experiments. The 4"Ca efflux was expressed as a rate coefficient; this represents the fraction of 45Ca leaving the gland per unit of time. The rate coefficient values were obtained using a PDP 8/L computer according to the following equation: x 100

ROt where RC = rate coefficient; C = radioactivity collected in the perfusate during collection interval t; and c = average "Ca content of the tissue during collection interval t. In some cases 45Ca efflux was expressed as a relative rate coefficient (A). This value represents the percentage increase or decrease in the rate coefficient obtained during stimulation of the gland as compared to the rate coefficient obtained before stimulation. In those experiments where catecholamine output was measured, samples from the perfusates were added to tubes (00 C) containing 10 jul. 1-9 N-HC1 per ml. perfusate. The catecholamine content of these samples was assayed by the trihydroxyindole fluorometric method described by Anton & Sayre (1962).

Subcellular fractionation of the adrenal medulla and isolation of chromaffin granules After the 4"Ca labelling phase, cortex-free glands were perfused for 60 min with Ca2+-free (EDTA) Locke solution as indicated above. The perfusion was then stopped and each adrenal medulla was homogenized in 4 vol. ice-cold 0-3 M sucrose (pH 7.0). The different subcellular fractions were isolated according to a technique

374

J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFAR6

previously described (Trifar6 & Dworkind, 1975). This method allows the separation of 6 fractions and a sediment which contains a relatively pure preparation of chromaffin granules as indicated by the enzyme determinations used as mitochondrial (monoamine oxidase), lysosomal (,8-glucuronidase), Golgi apparatus (galactosyl transferase) and plasma membrane (5'-nucleotidase) markers (Trifarr6 & Dworkind, 1975; Trifar6 & Duerr, 1976).

Chemicals The chemicals were obtained from the following sources: acetylcholine chloride, Welker Laboratories; disodium ethylenediamine tetra-acetate (EDTA) and LiCl, Fisher Scientific Company; choline chloride, The British Drug Houses Limited; sucrose (density gradient grade), Schwartz-Mann; 4"Ca (50 mc/m-muole) and [14C]sorbitol (200 mc/m-mole) New England Nuclear. D-600 (methoxyverapamil) was a generous gift from Drs Oberdorf and Sharma, Knoll A.C., Ludwigshafen. RESULTS

Effect of Na+-omission on the output of catecholamines from cQrtex-free glands In order to avoid the possible interference of the adrenal cortex in the 45Ca movement studies, all the experiments were carried out on cortexfree glands. Six experiments shown that substitution of extracellularNa+ by an osmotic equivalent amount of sucrose produced an increase in catecholamine output in the cortex-free glands, as has been observed when intact adrenal glands were used (Lastowecka & Trifar6, 1974). There was a clear increase in catecholamine output each time the perfusion fluid was switched from regular Locke to Na+-free Locke solution (Fig. 1); catecholamine output always returned to control levels after 10-15 min of perfusion with Na+-free Locke solution. Furthermore, as was the case with the intact glands, the responses declined with successive exposures to Na+-free solutions.

Desaturation curves of ["4C]sorbitol and 45Ca obtained in the adrenal medulla Fig. 2 compares desaturation curves for [14C]sorbitol, a marker for the extracellular space, and for "Ca obtained during perfusion with Ca2+-free Locke solution. The extracellular marker was almost completely washed out within 30 min of perfusion (Fig. 2); '5Ca was washed out more slowly than [L"Clsorbitol, indicating that radiocalcium was not confined only to the extracellular space, but that it had probably been taken up by the chromaffin cells. The miCa efflux curve was resolved in three different components by graphical analysis. These had a half time of 1-2, 4-1 and 33-5 min respectively. The slowest component presumably represents the washout of 45Ca from mainly intracellular sites. When the last component was drawn with values obtained from thirteen experiments, the plot was linear from 35 to 100 min, showing evidence that this phase repesents a

CALCIUM EFFLUX IN THE ADRENAL MEDULLA 375 single exponential. No further attempt was made either to analyse components 1 and 2 or to study the values obtained after the extrapolation to zero time of the data from the three different washout components. Under our experimental conditions we have assumed that all radioactivity present in the perfusates after 40 min is a unidirectional flux of 45Ca representing the washout of intracellular Ca2+.

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Fig. 1. Effect of Na+ omission on the output of catecholamines from cortex-free glands. Each bovine adrenal gland was Perfused alternately with Locke solution (LI) and Na+-free Locke solution (8) for periods of 27 and 3 min respectively. The glands were perfused at room temperature (250 C) with a flow rate of 10 ml./min. The perfusing solutions were gassed with a mixture of 5 0CO2 in 02 Samples were collected from the perfusates at 1 min intervals; these were assayed for catecholamine content as indicated in the Methods. The vertical bars represent the mean + S.E. of mean of six different experiments.

Effect of Na+ omission on the 45Ca efflux In order to test the effect of Na+ deprivation on "5Ca efflux, adrenal glands, previously labelled with 45Ca, were washed out with Ca2+-free Locke solution for 60 min and then they were exposed to a Na+-free, Ca2+-free Locke medium for an additional period of 10 min. As

376 J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFAR6 illustrated in Fig. 3, Na+ omission produced a sustained decrease in the rate coefficient of 45Ca efflux (Table 1). Fig. 3 also shows the increase in catecholamine output induced by Na+ deprivation. 100 r

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Fig. 2. Desaturation curves of [14C]sorbitol and 45Ca obtained in the adrenal medulla. Bovine adrenal glands were perfused for 25 min with Ca2+-free Locke solution containing 2 mM-EDTA; perfusion was then continued for 5 min with the same Ca2+-free solution but devoid of EDTA. This was followed by a 4 min period of perfusion with the same solution in which the glands were exposed to either 45Ca (@) 5 ptc/ml. (specific activity: 2-27 ,uc/n-mole) or to [14C]sorbitol (Q) 1 scp/ml. (specific activity: 200 ,uc//zmole). After this labelling phase, the perfusion was continued for 60 mmn with Ca2+-free Locke solution containing 2 mM-EDTA. All perfusing solutions were gassed with a mixture of 5% CO2 in 02 and the final pH of the solutions was 7-2. The glands were perfused at room temperature (25° C) with a flow rate of 10 ml./min. Samples of the perfusates were collected for a period of 1 min at 1 or 2 min intervals. The radioactivity present in the samples was determined as indicated in the Methods. The desaturation curves of 45Ca and [14C]sorbitol represent the mean of thirteen and two experiments respectively.

In all these experiments, Na+ was substituted by an osmotically equivalent amount of sucrose and since it has been previously observed that the substitution of Na+ by choline also produces a rise in catecholamine output (Lastowecka & Trifar6, 1974), 45Ca efflux was measured under these

CALCIUM EFFLUX IN THE ADRENAL MEDULLA 377 conditions. Table 1 shows that the substitution of Na+ by choline also produces a decrease in the "6Ca rate coefficient. Furthermore, perfusion of three other glands with Ca2+-free (Li+) Locke solution for more than 10 min produced a decrease in the rate coefficient (- 44 + 8 %). This effect which was not clearly observed during the first 5 min of perfusion, was found consistently during the long exposures to Ca2+-free (Li+) Locke solution.

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Fig. 3. Effect of Na+ omission on the "Ca efflux. A bovine adrenal gland was perfused and labelled with 4"Ca as described in Fig. 2. After 60 min of perfusion with CaO+-free Locke solution (0), the perfusion was switched to a Na+-free, Ca2+-free solution (U) for 10 min. Then perfusion was continued with Ca2+-free Locke solution. The Figure shows the desaturation curve of "Ca, the rate coefficient of the "Ca effliux and the catecholamine output. Similar results were obtained in three other experiments. Other conditions were as described in Fig. 2.

Similar results on the 45Ca efflux have been recently obtained with medullary slices, although in this case the Ca2+-free solution contained 3-6 mM-Mg2+ and the catecholamine output was not measured (Rink & Baker, 1975). Therefore, the present results demonstrate the requirement for external Na+ of the "Ca efflux into Ca2+-free Locke solution.

378 J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFAR6 Furthermore, although Na+ fluxes have not been measured in these experiments, the results also suggest the possibility that a Na+-Ca2+ exchange mechanism is present in the chromaffin cell. 1250 r

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Fig. 4. Effect of the omission of Na+ from the regular Locke solution on the efflux of "Ca in the presence or in the absence of methoxyverapamil (D-600). Two bovine adrenal glands were perfused and labelled with "Ca as described in Fig. 2. After 15 min of perfusion with Ca2+-free Locke solution, the glands were perfused with Locke solution for the following 45 min (E), the perfusion was then switched to a Na+-free Locke solution (-) for 10 min. Then perfusion was continued with Locke solution. Forty min after the experiment was started, methoxyverapamil (3 x 10-4 M) was added to the perfusion fluid of one (@) of the glands. This gland was perfused with solutions containing methoxyverapamil until the end of the experiment. The Figure shows the rate coefficients of the 45Ca efflux and the catecholamine outputs. Similar results were obtained with four other pairs of glands. Other conditions as described in Fig. 2.

CALCIUM EFFLUX IN THE ADRENAL MEDULLA

379

Effect of extracellular Ca2+ on the efflux of 45Ca during Na+ omission Since it has been shown that extracellular Ca2+ is not necessary for the release of catecholamines to be evoked by Na+ omission (Lastowecka & Trifaro, 1974), experiments tested whether the presence of Ca2+ (2.2 mM) in the perfusion fluid modified the decrease in 45Ca efflux observed during stimulation by Na+ deprivation. Adrenal glands were perfused and labelled with "5Ca as described in the Method section; they were washed out by perfusion with Ca2+-free Locke solution for 15 min and regular Locke solution for the following 45 min; then Na+-free (sucrose) Locke solution was introduced for 10 min. Fig. 4 shows that in contrast to the results presented in the preceding section, Na+ omission in the presence of extracellular Ca2+ induced a significant increase in 45Ca efflux (Table 1). This effect was significantly blocked when, in addition to Ca2+, 3 x 10-4 M TABLE 1. Effect of the substitution of Na+ by either sucrose or by choline on the 4"Ca efflux. Cortex-free adrenals which have been previously labelled with "Ca (5 juc/ml.) as indicated in the Methods were perfused for 60 min with either Locke or Ca2+-free Locke solution. This was followed by perfusion with solutions in which Na+ substituted by either sucrose or choline. In all these experiments the concentration of Mg2+ in the extracellular fluid was 1-2 mm. When the effect of 3 x 1I0- M methoxyverapamil (D-600) was tested, the drug was added to the perfusion fluid 20 min before the removal of Na+. Further experimental details are described in the text Relative rate coefficient Condition (A %) -48 + 3t Na+-free, Ca2+-free (sucrose) Locke (n = 4)* -30+ 5 Na+-free, Ca2+-free (choline) Locke (n = 4) + 81± 15 Na+-free (sucrose) Locke (n = 5) +10± 4 Na+-free (sucrose) Locke + D 600 (n = 5) *

Number of experiments.

t Mean + s.E. of mean.

methoxyverapamil (D-600) was present in the perfusion fluid (Table 1). However, as demonstrated previously (Pinto & Trifaro, 1976) D-600 did not affect the increase in catecholamine release induced by Na+ deprivation (Fig. 4). Therefore the fact that D-600 decreased the "5Ca efflux under the above conditions would suggest that the enhanced "5Ca efflux is due to a Ca2+-Ca2+ exchange mechanism. Effect of Mg2+ on the "Ca efflux during Na+ deprivation In the absence or in the presence of 1-2 mM-MgCl2, Na+ deprivation induces the release of catecholamines from the adrenal medulla (Lastowecka & Trifaro, 1974). However, increasing the extracellular concentration of Mg2+ to 10 and 20 mm produced 56-4 + 6-1 and 93-2 + 2.3 / i6

PHY 269

380 J. AaUIRRE, J. E. B. PINTO AND J. M. TRIFARO (mean + s.xE. of mean) inhibition respectively of the release of catecholamines in response to Na+ omission. Furthermore, Mg2+ was equally active in blocking the effect of Na+ omission on catecholamie release either in the presence or in the absence of extracellular Ca2+ (Lastowecka & Trifar6, 1974). B Ca2+-free, Mg2&-free Locke * Ca2+-free, Mg2+-free, Na+-free Locke

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Fig. 5. Effect of Na+ omission on the 4"Ca efflux in the absence of extracellular Mg2+. A bovine adrenal gland was perfused and labelled with "RCa as described in Fig. 2. After 60 min of perfusion with Ca2+-free Locke solution containing 2 mM-EDTA (j), the perfusion was continued with Na+free, Ca2+-free (EDTA), Locke solution (@) for 10 min. Then perfusion was switched back to Ca2+-free (EDTA), Locke solution. The Figure shows the desaturation curve of 45Ca and, in the inset, the rate coefficient of the 4"Ca efflux and the catecholamine output are represented. Similar results were obtained in nine other experiments. Other conditions were as described in Fig. 2.

In the presence of 20 mM-MgCl2, the decrease in the '45Ca rate coefficient produced by the removal of Na+ from the extracellular environment was similar to that obtained in the presence of 1-2 mM-MgCl2 (Table 2). However, in the absence of extracellular Mg2+, Na+ deprivation produced an unexpected effect on the efflux of 45Ca. Under these conditions, Na+

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381 omission induced a sharp and transient increase in the rate coefficient of the 45Ca efflux (Fig. 5, Table 2). The maximum increase was usually detected 2 min after the introduction of the Na+-free solution and the 45Ca efflux returned to basal values approximately 5-10 min after the beginning of the stimulation (Fig. 5). The time course of the "6Ca efflux seems to be similar to the secretary response to Na+ omission (Fig. 5).

CALCIUM EFFLUX IN THE ADRENAL MEDULLA

TABLE 2. Effect of Mg2+ on the 45Ca efflux evoked by Na+ omission. After 60 min of perfusion with Ca2+-free solution, the glands were stimulated by Na+ deprivation in the absence or in the presence of either 1P2 or 20 mM-MgCl2. Other conditions were as described in Table 1 Relative rate coefficient (A %) Condition -48 ± 3t Na+-free, Ca2+-free Locke + 12 mM-Mg2+ (n =4) -52 + 6 Na+-free, Ca2+-free Locke + 20 mMIMg2+ (n = 7) + 386 + 38 Na+-free, Ca2+-free Locke (n = 10) + 365 +25 Na+-free, Ca2+-free Locke + D-600 (n = 3) *

t

Number of experiments. Mean + s.E. of mean.

However, since collection of samples was at 1 min intervals, it was not possible to determine whether or not the peak in the "6Ca efflux preceded that of the output of catecholamines. Furthermore, as expected, methoxyverapamil (D-600) was unable to block this increase in the 45Ca efflux induced by the omission of Na+ from the extracellular fluid (Table 2). These results would suggest that under this experimental condition, that is, in the absence of extracellular Mg2+, Na+ deprivation induces the release of intracellular Ca2+ which might be the result of an increase in free Ca2+ within the cytosol. Furthermore, the results of two experiments in which Na+ was replaced by choline instead of sucrose showed 45Ca efflux increases of 218 and 224 % respectively. We have reported previously that there was a negative correlation between the logarithm of the extracellular concentration of Na+ and catecholamine output from the adrenal medulla (Lastowecka & Trifar6, 1974). Thus, experiments tested whether there is any relationship between the Na+ concentration in the perfusion fluid and the relative increase in the "6Ca efflux. The results showed (Fig. 6, left-hand side) that there was no difference in the time course of the 45Ca efflux in response to the presence of three different concentrations of Na+ in the perfusion medium. However, the results of these experiments suggested that a relationship might exist between the extracellular concentration of Na+ and the increase in 45Ca efflux. Results from nine other experiments revealed that a significant negative correlation existed between the logarithm of the extracellular x6-2

382 J. AGUIRRE, J. E. B. PINTO AND J. M. TRIPARO Na+ concentration and the maximum relative rate coefficient of the "Ca efflux (Fig. 6, right-hand side). It should also be pointed out that the extrapolation of the computer fitted regression line to normal extracellular Na+ value showed a dissociation between the theoretical 4"Ca basal efflux value and the experimental data. Although we have no precise explanation for this difference, it is possible that a small loosely bound 45Ca pool, probably of extracellular origin, may be responsible for this effect.

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Therefore, these results on the 45Ca efflux obtained with the graded substitution of Na+ by sucrose are quite similar to those in which catecholamine outputs were measured. The 45Ca released from the adrenal medulla during Na+ deprivation was 3-2 + 08 x 105 ct/min (n = 7), a figure which represents 5-6 % of the total 45Ca present in the gland at the time of stimulation. An adrenal

CALCIUM EFFLUX IN THE ADRENAL MEDULLA 383 medulla of an average weight (5.2 + 04; n = 55) contained 58+6 x 105 ct/min of 45Ca (n = 8) after 60 min of perfusion. Due to the small percentage of 45Ca released during Na+ deprivation, subcellular fractionation studies of the adrenal medulla showed no significant differences in the distribution of 45Ca between control and glands stimulated by Na+ deprivation (Fig. 7). This is also why efflux curves based on the "Ca efflux collected with the perfusates rather than was left in the tissues were used to express the results. Furthermore, no significant differences were observed in the subcellular distribution of catecholamines between control and glands stimulated by Na+ deprivation (Fig. 7). This was due to the fact that 3-3 + 0*5 (n = 13) #moles of catecholamines are released from the chromaffin granules during 10 min of exposure to a Na+-free solution. This figure is smaller than the standard error (5-7 #mole) obtained after averaging the catecholamine content of the different granule preparations (fraction no. 7 in Fig. 7). In addition, the results show that although all subcellular fractions were labelled with 45Ca, the mitochondrial fraction contained the largest percentage of ratioactive Ca. This seems to be in agreement with the subcellular distribution of "5Ca obtained in experiments with intact perfused glands (Winkler, Schopf, Hdrtnagl & Hdrtnagl, 1972). Therefore, it is possible that the mitochondrial fraction is the origin of the 45Ca found in the effluents during Na+ omission in the absence of extracellular Mg2+. The chromaffin granules can be discarded as the origin of the increased "5Ca efflux since, knowing the total amount of catecholamines released during Na+ omission and the ratio of 45Ca to catecholamines in the granules (7.2± 11 x 102 ct/min 45Ca/#mol catecholamine; n = 11), it was possible to estimate the amount of 45Ca released from chromaffin granules. Even on the assumption that, under these conditions, that is, in the absence of extracellular Mg2+, the total 45Ca present in the granules leaves the gland along with the catecholamines during release induced by Na+ omission, the granular calcium would contribute with 4 % or less to the total 45Ca present in the effluents.

Effect of Ca2+ re-introduction on the 45Ca efflux Douglas & Rubin (1961) have shown that, in the cat adrenal medulla, calcium releases catecholamines when it is reintroduced into the perfusion fluid after the glands have previously been exposed to Ca2+-free medium containing EDTA. They have suggested that this effect was due to an inward movement of Ca2+ leading to the accumulation of an excess of Ca2+ at some specific site in the chromaffin cell which triggers the release of stored catecholamines. Furthermore, during the course of the experiments mentioned in the preceding sections, an increase in "Ca efflux was observed upon switching the perfusion from Ca2+-free (EDTA)

J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFAR6

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Fig. 7. The subcellular distribution in the adrenal medulla of catecholamines and 46Ca. Six bovine glands were perfused and labelled with "Ca as described in Fig. 2. The glands then perfused for 60 min with Ca2+-free (EDTA) Locke solution. Then perfusion was continued for 10 min with ) and with Ca+-free, Ca2+-free Locke solution in three of the glands ( Na+-free Locke solution in the other three glands (---). After this period of perfusion, each medulla was homogenized and the 'crude granule fraction' was obtained as described previously (Trifar6 & Dworkind, 1975). The 'crude granule fraction' was layered on top of a discontinuous sucrose density gradient. The polarities of the sucrose solutions used in the preparation of the gradient are indicated at the top of the Figure. The tube was centrifuged at 113,000 g for 70 min and six fractions and a sediment were separated from top to bottom. The fractions were analysed for catecholamines, 4"Ca, monoamine oxidase and 8l-glucuronidase as described in the Methods. p-HPHA is the abbreviation for p-hydroxyphenylacetaldehyde.

CALCIUM EFFLUX IN THE ADRENAL MEDULLA 385 Locke solution to Locke (2.2 mM-Ca2+) solution. Therefore, experiments were also performed under our experimental conditions in order (a) to determine the magnitude of "Ca mobilization by the reintroduction of 600

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Fig. 8. Effect of methoxyverapamil (D-600) on the output of catecholamines and the "Ca efflux in response to Ca2+ reintroduction. Two bovine adrenal glands were perfused and labelled with "'Ca as described in Fig. 2. After 60 min of perfusion with Ca'+-firee Locke solution (s), the perfusion was continued for 10 min with regluar Locke solution (E3). Forty min after the experiment was started, methoxyverapamil (3 x 10-4 m) was added to the perfusion fluid of one (-) of the glands. This gland was perfused with solutions containing methoxyverapamil during the rest of the experiment. Samples of the effluent were collected and assayed for catecholamines and "Ca content as indicated in Methods. Catecholamine outputs and "Ca rate coefficients are illustrated at the top and bottom of the Figure respectively. Similar results were obtained with ten other glands. Other conditions were as described in Fig. 2.

J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFARO 386 Ca2+ into the perfusion fluid, and (b) to compare the 45Ca efflux induced by Ca2+ reintroduction with that produced by the omission of Na+ in the absence or in the presence of extracellular Mg2+. 150 o

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Fig. 9. Effect of Mg2+ on the output of catecholamines and the "Ca efflux in response to successive exposures to Na+ deprivation and Ca2+ reintroduction. A bovine adrenal gland was perfused and labelled with "Ca as described in Fig. 2. The gland was perfused for 60 min with Ca2+-free Locke solution (Q), and then the gland was perfused for three successive periods of 10 min each with Na+-free, Ca2+-free Locke (M), Ca2+-free Locke (E) and regular Locke ([) solutions respectively. Allsolutions contained 20 mrMgCl2. Samples of the effluent were collected and assayed for catecholamines and "Ca content as described in the Methods. The catecholamine output and the 'Ca rate coefficient are illustrated at the top and at the bottom of the Figure respectively. Other conditions were as described in Fig. 2.

CALCIUM EFFLUX IN THE ADRENAL MEDULLA 387 Fig. 8 illustrates one of these experiments which shows that calcium reintroduction for a period of 10 min produces a transient increase in the 45Ca efflux as well as a sharp release of catecholamines. The results obtained in eight experiments are shown in Table 3. Since Mg2+ (20 mM) inhibits the secretary response induced by Ca2+ reintroduction (Douglas & Rubin, 1961) we tested the effect of Mg2+ on the "5Ca efflux during this condition. The presence of Mg2+ blocked the release of catecholamines, but did not block the increase in the 45Ca efflux in response to Ca2+ reintroduction (Fig. 9 and Table 3). Furthermore, the contrasting effects of 20 mM-Mg2+ on the efflux of Ca2+ induced by either Na+ omission or Ca2+ reintroduction are also shown in Fig. 9. TABLE 3. Effect of Mg2+ and methoxyverapamil on the 45Ca efflux and catecholamine output induced by the reintroduction of Ca2+ into the perfusion fluid. After 60 min of perfusion with Ca2+-free Locke solution, the perfusion was continued for 10 min with Locke solution containing (a) 1-2 mM-MgCl2; (b) 20 mM-MgCl2 or (c) 1-2 MM-MgCl2 and 3 x 10-4 M methoxyverapamil (D-600). Samples of the effluents were collected and assayed for catecholamine and 46Ca content as indicated in the Methods. Other conditions were as described in Table 1 Relative rate coefficient (A %) + 1217 + 78* + 1378 ± 158

Condition Ca2+ reintroduction (n = 8)** Ca2+ reintroduction + 20 m-MMg2+ (n = 3) + 231 ± 27 Caa+ reintroduction + D-600 (n = 4) * Number of experiments.

Increased catecholamine output (A %) + 513 + 97 + 28+ 6 + 28 9

t Mean + s.E. of mean.

It has been shown that methoxyverapamil (D-600), a calcium antagonist, blocks the release of catecholamines from the adrenal medulla in response to stimulation by either acetylcholine or a depolarizing concentration of potassium (Pinto & Trifaro, 1976). Therefore the effect of D-600 (3 x 10-4 M) on both catecholamine release and 45Ca efflux in response to Ca2+ reintroduction was tested. Table 3 shows that D-600 was as powerful as 20 mM-Mg2+ in blocking catecholamine release. However, contrary to the effects of Mg2+ on 45Ca efflux, D-600 produced a significant decrease of the 4Ca efflux in response to Ca2+ reintroduction (Fig. 8 and Table 3). The transient peak in the efflux curve which remains even in the presence of D-600 could probably be explained as an ion exchange at superficial sites. These experiments would suggest that (a) 81 % of the increased 45Ca efflux observed during Ca2+ reintroduction may be due to a Ca2+-Ca2+ exchange mechanism, since this is the percentage of inhibition obtained

388 J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFAR6 in the presence of D-600, (b) the release of catecholamines in response to Ca2+ reintroduction requires only extracellular Ca2+ and (c) Mg2+ does not block Ca2+ entry and the inhibition of catecholamine release might be the result of a competition between Ca2+ and Mg2+ for an intracellular site. DISCUSSION

The present experiments have provided new information on the movement of calcium ions in the perfused adrenal medulla. The stimulation of the adrenal glands by Na+ deprivation induces release of catecholamines (Lastowecka, & Trifaro, 1974). Furthermore, in the presence or in the absence of extracellular Ca2+, the release induced by Na+ omission is by exocytosis because the amines released were accompanied by ATP and dopamine fl-hydroxylase (Lastowecka & Trifaro, 1974). Therefore, if in response to different stimuli (acetylcholine, high K+, Na+ deprivation) release by exocytosis would involve similar cellular and molecular mechanisms, we must assume that during Na+ omission there is an increase in intracellular Ca2+. The present results show that substitution of Na+ from the extracellular medium by either sucrose or choline increases the output of catecholamines but decreases the 4Ca efflux. These observations support the above suggestion of an increase in the concentration of intracellular Ca2+ during Na+ omission. This decrease in the "Ca efflux was observed only in the absence of extracellular Ca2+ and the most probable explanation for this effect is that it was due to the inhibition of the Na+-Ca2+ exchange as a result of the removal of Na+ from the extracellular environment. On the assumption of the existence of a Na+-Ca2+ exchange mechanism in the fi cell of the dog pancreas, the suggestion has been made that the increase in insulin release during Na+ omission might be the result of a decrease in the Ca2+ efflux following the removal of extracellular Na+ (Griffey, Conaway & Whitney, 1974). Our observations on the "Ca efflux in the adrenal medulla, another endocrine tissue, seem to support this

hypothesis. The existence of a Na+-dependent Ca2+ efflux was first observed in the squid axon and in the cardiac muscle (Baker & Reuter, 1975). In the squid axon the removal of Na+ from the extracellular space produced a decrease in the efflux of Ca2+ and the effect is fully reversible in a Ca2+-free medium (Baker, 1970). Similarly, the effect of Na+ omission on the efflux of 4Ca from the adrenal medulla is reversible provided that the glands are perfused with a Ca2-free solution. In the presence of extracellular Ca2+ the situation is different. Here the removal of Na+ induced a significant increase in the 45Ca efflux. A similar

CALCIUM EFFLUX IN THE ADRENAL MEDULLA 389 observation has been made recently on adrenal medullary slices incubated in vitro in the presence of extracellular Ca2+ (Rink & Baker, 1975). The increase in the "6Ca efflux under these conditions may be due to the presence in the adrenal medulla of a Ca2+-Ca2+ exchange mechanism similar to that described for the squid axon. Furthermore, under these experimental conditions, that is, in the absence of extracellular Na+, Ca2+ entry may be enhanced, since as in the squid axon (Baker & Reuter, 1975) a Na+-Ca2+ antagonism may also exist in the adrenal medulla. Therefore it may well be that Na+ deprivation removes a Na+-dependent component of the Ca2+ efflux, increasing at the same time Ca2+-Ca2+ exchange. This would produce a decrease in the net Ca2+ efflux in spite of an increased 45Ca efflux. The idea that part of the enhanced 4Ca efflux observed under the above conditions is due to a Ca2+-Ca2+ exchange mechanism is supported further by the observation that methoxyverapamil (D-600), a Ca2+ antagonist (Fleckenstein, 1971; Fleckenstein, Grun, Tritthart & Byon, 1971; Mayer, van Breemen & Casteels, 1972), produced a significant decrease in the 45Ca efflux. This later finding would also suggest that Ca2+ entry in the adrenal medulla is at least, in part, through a channel of similar properties to that described for the squid axon as the 'late calcium channel'. D-600 has been also shown to block the late phase of Ca2+ entry in the squid axon (Baker, Meves & Ridgway, 1973). In spite of all these effects on the "Ca efflux, catecholamine release in response to Na+ deprivation remained unchanged. This is in agreement with previous observations showing that Na+ omission induces catecholamine release either in the absence or in the presence of extracellular Ca2+ (Lastowecka & Trifaro, 1974) as well as in the presence of D-600 (Pinto & Trifaro, 1976). Increasing the extracellular concentration of Mg2+ blocks the release of catecholamines in response to Na+ deprivation. However, the decrease in the 45Ca efflux produced by Na+ omission was similar when the extracellular concentration of Mg2+ was either 1-2 or 20 mm. Moreover, when adrenal glands were perfused with a Na+-free solution devoid of Mg2+ a different phenomenon was observed, a fourfold increase in 45Ca efflux. This effect does not seem to be due to the replacement of Na+ by sucrose in the perfusion fluid since it was also observed when Na+ was substituted by choline. In addition, this increased 45Ca efflux was not modified by the presence of D-600 in the perfusion fluid, thus indicating the possible intracellular origin of the "5Ca. It seems therefore that in the absence of Mg2+, the increase in intracellular Ca2+ that might be produced by Na+ omission is followed by its extrusion from the chromaffin cell. This effect does not seem to be the result of an increased permeability of the cell membrane during Na+ omission in the absence of extracellular Mg2+ for it has been previously shown that, under similar conditions, the cell

390 J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFAR6 membrane permeability, evaluated by the efflux of lactate dehydrogenase (a marker for the cytosol), remained unchanged (Lastowecka & Trifaro, 1974). This effect is possible due to the fact that Mg2+ may be necessary for the retention or uptake of Ca2+ into some subcellular organelles. This idea is supported by the observation of Poisner & Hava (1970) on the Mg2+ dependency in the mechanism of uptake and binding of "Ca into adrenal medullary microsomes. A similar explanation for the decrease in the "Ca efflux observed in the submandibular gland of the cat in response to a high Mg2+ concentration has been previously advanced (Nielsen & Petersen, 1972). However, the mitochondrion might be another target for the intracellular action of Mg2+ since it is known that this divalent cation is necessary for the active calcium uptake by this organelle (Lehninger, 1970; Haugaard, Haugaard & Lee, 1969; Poisner & Hava, 1970). Moreover, recent evidence suggests that in nerve terminals, mitochondria might play a role in transmitter release by participating in the regulation of intracellular free Ca2+ (Almaes & Rahamimoff, 1975). If Na+ omission is responsible for the observed increase in 45Ca efflux in the absence of extracellular Mg2+, some kind of relationship might exist between the extracellular Na+ concentration and this increased 45Ca efflux. This proved to be the case since the results reported here show that a graded substitution of extracellular Na+ produced a proportional increase in the 45Ca efflux. A similar relationship between catecholamine output and extracellular Na+ concentration has been previously obtained (Lastowecka & Trifaro, 1974). The return to normal values of the enhanced "Ca efflux observed during Na+ deprivation may be the result of a progressive decline in the concentration of free cytoplasmic Ca2+. Furthermore, if this increase in cytoplasmic calcium were responsible for the catecholamine release observed during Na+ omission, its decline may be the reason why during Na+ omission the high catecholamine output is not maintained. The inactivation of the 'late calcium channel' by a prolonged depolarization has been suggested as an explanation for the decrease in catecholamine output observed during high K+ stimulation (Baker & Rink, 1975), This is obviously not the case during Na+ omission since the effects on 45Ca efflux and on catecholamine release were observed in the absence of extracellular Ca2 . The results presented here also indicate that a very small amount (5-6 %) of the total 4"Ca present in the gland is released at the time of stimulation by Na+ omission. For this reason, the exact intracellular source of the radiocalcium found in the effluent under this condition is uncertain at the present time. However, the chromaffin granules may be discarded as the main source of the 45Ca appearing in the perfusates during Na+ omission in the absence of extracellular Mg2+ because in the

CALCIUM EFFLUX IN THE ADRENAL MEDULLA 391 presence of 1P2 mM-Mg2+, Na2+ deprivation induced a decrease in the 46Ca efflux with a concomitant increase in catecholamine output. Furthermore, from data obtained in subcellular fractionation studies, it may be concluded that chromaffin granules would contribute less than 4 % to the total amount of 46Ca present in the effluent during Na+ omission. Therefore, it seems that Conditions which produce an increase in the [Na+]1/ [Na+]o ratio will favour the accumulation of intracellular Ca2+ which may be necessary to trigger hormone and transmitter release. The [Na+],/ [Na+]o ratio can be increased either by removing extracellular Na+ or by increasing intracellular Na+ as a result of blocking the Na+ plimp either by K+ removal or by ouabain treatment. All these conditions have been shown to increase hormone release (Trifaro, 1977). The presence of a Ca2+-Ca2+ exchange mechanism in the adrenal medulla may be suggested not only from the results obtained during Nat omission in the presence of extracellular Ca2+, but also from the data obtained during the reintroduction of Ca2+ into the perfusion fluid. It is well known that plasma membranes requires Ca2+ in order to maintain the relative impermeable state. Thus, when Ca2+ is withdrawn from the extracellular medium the permeability of the plasma membrane is enhanced. This also seems to be the case in the adrenal medulla where Ca2+ reintroduction into the perfusion fluid induces a significant discharge of catecholamines (Douglas & Rubin, 1961). This effect has been interpreted as due to a rapid penetration of Ca2+ through a highly permeable plasma membrane (Douglas, 1968). Furthermore, increasing the extracellular concentration of Mg2+ blocked the effect of Ca2+ reintroduction on catecholamine release (Douglas, 1968). The present results on the release of catecholamines during Ca2+ reintroduction and its inhibition by Mg2+ confirm the above findings. Moreover, our results also show that Ca2+ reintroduction produced a vigorous release of '6Ca. However, and contrary to the observation on catecholamine release, Mg2+ was unable to decrease this enhanced 4"Ca efflux. In connexion with this latter observation is the finding that the increase of the extracellular concentration of Mg2+, even to 100 mM, did not modify the Ca2+-Ca2+ exchange component of crustacean muscle fibres (Ashley, Ellory & Hainaut, 1974). On the other hand, methoxyverapamil (D-600) not only blocked catecholamine release but it also produced 81 % inhibition ofthe increase in 45Ca efflux in response to Ca2+ reintroduction. Methoxyverapamil has been also shown to inhibit the release of vasopressin and oxytocin from the neurohypophysis (Dreifuss, Grau & Nordmann, 1973; Russell & Thorn, 1974; Thorn, 1974) and the release of catecholamines from the adrenal medulla in response to either acetylcholine or high K+ stimulation (Pinto & Trifaro, 1976). All these results allow us to suggest the following interpretations: (a) that

392 J. AGUIRRE, J. E. B. PINTO AND J. M. TRIFAR6 this vigorous increase in 4Ca efflux occurs in a large proportion in exchange with intracellular Ca2+; (b) the presence of a 'late calcium channel' in the adrenal medulla; (c) that Mg2+ does not interfere with the entry of Caa2+ into the chromaffin cell, although this point deserves further experimental work, and (d) that Mg2+ blocks the catecholamine release induced by Ca2+ reintroduction by competing with Ca2+ for an intracellular site. Previous published observations have also suggested an intracellular site as the place of competitition between Ca2+ and Mg2+ during the release reaction (Miledi, 1973; Kanno, Cochrane & Douglas, 1973; Lastowecka & Trifar6, 1974). This work was supported by grants from the Medical Research Council of Canada. J. E. B. Pinto holds a Postdoctoral Fellowship from the M.R.C. of Canada and J. Aguirre holds a Postdoctoral Fellowship from C.U.S.O. We are grateful to Miss C. Shorten for her skilled technical assistance, and to Mr R. Cardinal for helping us during the initial experiments and in the preparation of the computer programs. REFERENCES ALMA S, E. & RAHaIamoFF, R. (1975). On the role of mitochondria in transmitter release from motor nerve terminals. J. Phyeiol. 248, 285-306. ANTON, A. H. & SAYmE, D. F. (1962). A study of the factors affecting the aluminum oxide trihydroxyindole procedure for the analysis of catecholamines. J. Pharmac. exp. Ther. 138, 360-371. AShLEY, C. C., ELLORY, J. C. & HAiNAUT, K. (1974). Calcium movements in single crustacean muscle fibres. J. Phy8iol. 242, 255-272. BAKER, P. F. (1970). Sodium-calcium exchange across the nerve cell membrane. In Calcium and Cellular Function, ed. CUTHBERT, A. W., pp. 96-107. London: Macmillan. BAKER, P. F., MEVES, H. & RIDGWAY, E. B. (1973). Calcium entry in response to maintained depolarization of squid axons. J. Phyeiol. 231, 527-548. BAKER, P. F. & REUTER, H. (1975). In Calcium Movement in Excitable Cells, pp. 1-100. Oxford: Pergamon. BAKR, P. F. & RniN, T. J. (1975). Catecholamine release from bovine adrenal medulla in response to maintained depolarization. J. Physiol. 253, 593-620. DouGLAs, W. W. (1968). Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. Br. J. Pharmac. Chemother. 34, 451-474. DouGLAs, W. W. & RUBIN, R. P. (1961). The role of calcium in the secretary response of the adrenal medulla to acetylcholine. J. Phyeiol. 159, 40-57. DREIFuSS, J. J., GRAu, J. D. & NORDMANN, J. D. (1973). Effects on isolated neurohypophysis of agents which affect the membrane permeability to calcium. J. Physiol. 231, 96-98P. FLAcKENsTEn, A. (1971). Specific inhibitors and promoters of calcium action in the excitation-contraction coupling of heart muscle and their role in the prevention of production of myocardial lesions. In Calcium and the Heart, pp. 135-188. London and New York: Karger. FIcxENsTEwN, A., GRuN, G., TRIrTHART, H. & BYON, K. (1971). Uterusrelaxation durch hechaktire Ca++ antagonistische Hemmstoffe der elecktromechanischen Koppelung wie Isoptin (Verapamil; Iproveratril), Substanz D600 und Segotonin (Prenylamin). Klin. Wschr. 49, 32-41.

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GRIFFEY, M. A., CONAWAY, H. H. & WHITNEY, J. E. (1974). Insulin secretion induced by Na+-deprivation. Endocrinology 95, 1469-1472. HAUGAARD, N., HAuGAARD, E. S. & LEE, N. H. (1969). Role of magnesium, phosphate and ATP in the regulation of calcium uptake by rat-liver mitochondria. Proc. K. ned. Akad. Wet. B 72, 1-15. KANNo, T., CocHRANE, D. E. & DouotAs, W. W. (1973). Exocytosis (secretory granule extrusion) induced by injection of calcium into mast cells. Can. J. Physiol. Pharmacol. 51, 1001-1004. LASTOWECKA, A. & TRIFAR6, J. M. (1974). The effect of sodium and calcium ions on the release of catecholamines from the adrenal medulla: sodium deprivation induces release by exocytosis in the absence of extracellular calcium. J. Phyeiol. 236, 681-70. LEHNINGER, A. L. (1970). Mitochondria and calcium ion transport. Biochem. J. 119, 129-138. MAYER, C. J., VAN BREEMEN, C. & CASTEELS, R. (1972). The action of Lanthanum and D600 on the calcium exchange in the smooth muscle cells of the guinea-pig taenia coli. Pfliuger8 Arch. ge8. Phy8iol. 337, 333-350. MEMDI, R. (1973). Transmitter release induced by injection of calcium ions into nerve terminals. Proc. B. Soc. B 183, 421-425. NEILSEN, S. P. & PETERSEN, 0. H. (1972). Transport of calcium in the perfused submandibular gland of the cat. J. Phyeiol. 223, 685-697. PINTO, J. E. B. & TRIFAR6, J. M. (1976). The different effects of D600 (methoxyverapamil) on the release of adrenal catecholamines induced by acetylcholine, high potassium or sodium deprivation. Br. J. Pharmac. 57, 127-132. PINTO, J. E. B., TRIFAR6, J. M. & CARDINAL, R. (1975). Increase in 4"Ca efflux from bovine adrenal medulla during catecholamine release induced by Na+ deprivation. VIth Int. Cong. Pharmac., Helsinki, p. 989. POISNER, A. M. & HAVA, M. (1970). The role of ATP and ATPase in the release of catecholamines from the adrenal medulla. 4. ATP-activated uptake of calcium microsomes and mitochondria. Molec. Pharmacol. 6, 407-415. RINK, T. J. & BAKER, P. F. (1975). The role of the plasma membrane in the regulation of intracellular calcium. In Calcium Transport in Contraction and Secretion, ed. CARAFOLI, E. et al., pp. 235-242. Amsterdam: North-Holland. RUSSELL, J. T. & THORN, N. A. (1974). Calcium and stimulus-secretion coupling in the neurophypophysis. II. Effects of lanthanum, a verapamil analogue (D-600) and prenylamine on 4"Calcium transport and vasopressin release of isolated rat neurophysis. Acta endoer., Copenh. 76, 471-487. SMITH, A. D. & WAXKLER, H. (1972). Fundamental mechanism in the release of catecholamines. In Handbook of Experimental Pharmacology, vol. 23, ed. BLsHuxo, H. & MUSCHOLL, E., pp. 538-617. New York: Springer-Verlag. THORN, N. A. (1974). Role of calcium in secretary processes. In Secretory Mechanism of Exocrine Glands, ed. THORN, N. A. & PETERSEN, 0. H., pp. 305-326. Copenhagen: Munksgaard. TRIFAR6, J. M. (1977). Common mechanisms of hormone secretion. A. Rev. Pharmac. (in the Press). TRIFAR6, J. M. & DuERR, A. C. (1976). Isolation and characterization of a Golgirich fraction from the adrenal medulla. Biochim. biophys. Acta 421, 153-167. TRIFAR6, J. M. & DWORKIND, J. (1975). Phosphorylation of the membrane components of chromaffin granules: synthesis of diphosphatidylinositol and presence of phosphatidylinositol kinase in granule membranes. Can. J. Physiol. Pharmacol. 53, 479-492.

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TRIFAR6, J. M., POISNER, A. M. & DOUGLAS, W. W. (1967). The fate of the chromaffin granule during catecholamine release from the adrenal medulla. 1. Unchanged efflux of phospholipid and cholesterol. Biochem. Pharmac. 16, 2095-2 100. WINKLER, H., SCHOPF, J. A. L., HORTNAGL, H. & HORTNAGL, H. (1972). Bovine adrenal medulla: subcellular distribution of newly synthesized catecholamines, nucleotides and chromogranins. Naunyn-Schmiedeberg8 Arch. Pharmacol. 273, 43-61.

Calcium movements during the release of catecholamines from the adrenal medulla: effects of methoxyverapamil and external cations.

371 J. Phyeiol. (1977), 269, pp. 371-394 With 9 text-ftguree Printed in Great Britain CALCIUM MOVEMENTS DURING THE RELEASE OF CATECHOLAMINES FROM TH...
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