J. Physiol. (1969), 2, pp. 797-85 797 With 7 text-ftgurem Printed in Great Britain SODIUM IONS AND THE SECRETION OF CATECHOLAMINES By P. BANKS, ROSEMARY BIGGINS, R. BISHOP, B. CHRISTIAN AND N. CURRIE From the Department of Biochemistry, Sheffield University (Received 23 September 1968) SUMMARY 1. Perfusion of bovine adrenal glands with a potassium-free Tyrode solution results in an increased spontaneous release of catecholamines and an increased response to stimulation with carbamylcholine. 2. Perfusion with a Tyrode solution containing 7 mm-kcl causes a marked secretion ofcatecholamines and a subsequent inhibition ofsecretion in response to stimulation with carbamylcholine. 3. Perfusion with sodium-free media abolishes or severely reduces the secretory response of the gland to carbamylcholine and to 7 mm-kcl although the basal secretion shows an initial rise. 4. Perfusion with sodium-deficient media permits some retention of the secretory response. 5. A possible role for intracellular sodium ions in the secretion of catecholamines is discussed. INTRODIUCTION When Douglas & Rubin (1961) described the involvement of Ca2+ ions in the secretory response of the adrenal medulla they also observed that perfusion with a K+-free medium enhanced the response, whilst replacement of NaCl by sucrose had little effect at first but tended to reduce the response as time went on. More recently, Banks (1967) found that the addition of ouabain to the perfusing Tyrode solution increased both the basal secretion and the secretory response to injected carbamylcholine. At about the same time, Milner & Hales (1967, 1968) observed that ouabain stimulated the release of insulin from slices of pancreas whilst the omission of sodium from the medium depressed it. As these studies on the secretory response of the adrenal medulla and the /8 cells of the pancreas were in progress, Baker and his colleagues were investigating a sodium-dependent influx of Ca2+ ions into the axons of Loligo forbesi and Maia squinado (Baker, Blaustein, Hodgkin & Steinhardt, 1967a; Baker, Blaustein, Manil & Steinhardt, 1967b; Baker & Blaustein, 1968). This Ca2+ influx was
798 P. BANKS AND OTHERS enhanced by increasing the intracellular concentration of sodium or by lowering the extracellular concentration of sodium and was unaffected by ouabain. The present experiments show that the Ca2+-dependent secretory response of the adrenal medulla fails when sodium is omitted from the perfusing solution and is enhanced under conditions which might be expected to increase the intracellular concentration of sodium. METHODS Bovine adrenal medullae were perfused and stimulated in vitro as described by Banks (1966). The Tyrode solution had the composition NaCl, 137 mm; KCI, 2-7 mm; MgCl2, 41 mm; CaCi2, 1V8 mm; NaHCO3, 11F9 mm; NaH2PO4, 4 mm; glucose, 5-6 mm and was gassed with a mixture of 95 % 2 + 5 % C2. In sodium-free or sodium-deficient solutions, NaCl was replaced by isosmotic amounts of LiCl, choline chloride or sucrose whilst NaHCO, and NaH2PO4 were replaced by the corresponding potassium salts unless stated otherwise. In potassium-free media KCI was replaced by NaCl. The catecholamine content of the perfusate was estimated by the method of Euler & Hamberg (1949). RESULTS The spontaneous secretion of catecholamines by the adrenal medulla showed a progressive fall during 21 hr periods of perfusion with Tyrode solution. However, the responses evoked by successive injections of carbamylcholine did not differ greatly, although injections made during the last 11 hr were usually somewhat less effective than those made during the first 1 hr. Perfusion with K+-free media. Figure 1 shows that 15 min after changing to perfusion with a K+-free Tyrode solution the basal secretion of catecholamines was higher than during the periods of perfusion with normal Tyrode solution. In addition, the response to stimulation by carbamylcholine was greater during the period of perfusion with the K+-free medium than in either of the control periods. Perfusion with K+-enriched media. When the concentration of potassium in the perfusing solution was increased to 7 mm at the expense of an equivalent amount of NaCl, there was a marked secretion of catecholamines which declined to normal values within 7 min (Fig. 2). Thereafter the response to stimulation with carbamylcholine was almost abolished until perfusion with normal Tyrode solution was resumed. In the presence of 15 mm potassium the secretory response was diminished only slightly. Perfusion with Na+-free media. Secretion in response to injections of carbamylcholine could be abolished by perfusion with sodium-free media (Fig. 3). These media were made by replacing NaCl by LiCl (Fig. 3B) or choline chloride (Fig. 3A) and substituting the corresponding potassium salts for NaHCO3 and NaH2PO4 (Fig. 3). The secretory response was
SODIUM AND CATECHOLAMINE SECRETION 799 restored, albeit in diminished form, after perfusion with Tyrode solution had been recommenced. In similar experiments where sucrose replaced NaCl, the secretory response was not abolished over a period of 1i hr although it was considerably reduced (Fig. 4). The response was not improved following the recommencement of perfusion with Tyrode solution. 4~~~~~~~~~~~~~ Fig. l. Perfusion of a bovine adrenal gland with a potasium-free Tyrode solution. The gland was perfused with Tyrode solution for 3 min before the beginning of the experiment. The open bar indicates perfusion with Tyrode solution and the filled bar indicates perfusion with potassium-free Tyrode solution. Carbamylcholine (.5 ml. 1 mm) was injected at the times indicated by the arrows. The perfusion rate was approximately 2 ml./min and the temperature was maintained at 37. The perfusing solutions were gassed with a mixture of 95 % 2 + 5 % C2. 3 4 4-2- 1 2 3 4 5 6 Fig. 2. Perfusion of a bovine adrenal gland with a potassium-enriched Tyrode solution. The open bar indicates perfusion with Tyrode solution and the filled bar indicates perfusion with a Tyrode solution in which 7 mm-nacl was replaced by 7 mm-kcl. Other conditions were as described in Fig. 1. On switching from Tyrode solution to sodium-free solutions, the basal secretion of catecholamines invariably increased at first but subsequently fell to normal values. The retention of 34 mm-nacl in LiCl-based solutions allowed a distinct secretory response to carbamylcholine to be demonstrated about i hr after 51 Phy. 2
8 P. BANKS AND OTHERS 4 4 3 ~~~~~~~~~~~~~A b 1 L r frmi rm P fm:la ce 1 73 115 152 3 - m is 5 1 15 Fig. 3. Perfusion of bovine adrenal glands with a sodium-free Tyrode solution. A. The open bar indicates perfusion with Tyrode solution and the filled bar indicates perfusion with a Tyrode solution in which all the NaCl was replaced by choline chloride. B. The open bar indicates perfusion with Tyrode solution and the filled bar indicates perfusion with a Tyrode solution in which all the NaCl was replaced by LiCl. In both sodium-free solutions NaHCO8 and NaH2PO4 were replaced by the corresponding potassium salts. Other conditions were as described in Fig. 1. 6 5 4 3 2 t 6 + + Jr ffi~~~~~~~~~~~ff 2 5 1 15 Fig. 4. Perfusion of a bovine adrenal gland with a sodium-free Tyrode solution. The open bar indicates perfusion with Tyrode solution and the filled bar indicates perfusion with a Tyrode solution in which all the NaCl was replaced by sucrose and NaHCO3 and NaH2PO4 were replaced by the corresponding potassium salts. Other conditions were as described in Fig. 1.
SODIUM AND CATECHOLAMINE SECRETION 81 changing from perfusion with Tyrode solution (Fig. 5A). Even after 11 hr of perfusion with this Na+-deficient solution, a small response was observed. When 68 mm-nacl was retained, very distinct responses to stimulation were obtained at both 2 I and 1I 2 hr after changina to the Na+-deficient solution (Fig. 5B). 3-4 ~~~~~~~~~~~~~~~~A 2, 2 5 1 5) 3 o)2 24h I 2 5 1 Fig. 5. Perfusion of bovine adrenal glands with sodium-deficient Tyrode solutions. Open bars indicate perfusion with Tyrode solution. A. Filled bar indicates perfusion with a Tyrode solution in which all but 34 mm-nacl was replaced by LiCl. B. Filled bar indicates perfusion with a Tyrode solution in which all but 68 mm- NaCl was replaced by LiCl. In both sodium-deficient solutions NaHCO3 and NaH2PO4 were replaced by the corresponding potassium salts. Other conditions were as described in Fig. 1. Perfusion with K+-enriched, Na+-free media. Raising the potassium concentration to 7 mm at the expense of LiCl during perfusion with a solution in which LiCl replaced NaCl failed to increase the secretion of catecholamines (Fig. 6). Perfusion with Na+-deficient media. In the experiments described above, 51-2
82 P. BANKS AND OTHERS which involved the use of sodium-free solutions or solutions containing 34 or 68 mm sodium chloride, NaHCO3 and NaH2PO4 were replaced by KHCO3 and KH2PO4. Thus in those solutions the potassium concentration was 15- mm compared with 2-7 mm in Tyrode solution. Although control experiments indicated that the secretory response was essentially unaffected by potassium concentrations of 15 mm in the perfusing solutions, confirmation of the results obtained in sodium-free media was 4 - f r 3z2,- Ilte -~ 4 o V 2 5 7 9 1 1 35 Fig. 6. Perfusion of a bovine adrenal gland with a potassium-enriched sodiumfree Tyrode solution. The open bar indicates perfusion with Tyrode solution; the filled bar indicates perfusion with a Tyrode solution in which all the NaCl was replaced by LiCl and the hatched bar indicates perfusion with a LiCl-containing, sodium-free Tyrode solution in which 7 mm of the LiCl was replaced by KCl. In both sodium-free media NaHCO, and NaH2PO4 were replaced by the corresponding potassium salts. Other conditions as described in Fig. 1. 5 o 4 I2-3 v 35 so 1 1 so Fig. 7. Perfusion of a bovine adrenal gland with a sodium chloride-free Tyrode solution. Open bars indicate perfusion with Tyrode solution and the filled bar indicates perfusion with a Tyrode solution in which all the NaCl was replaced by LiCl but which contained normal amounts of NaHCO8 and NaH2PO4. Other conditions as described in Fig. 1. sought using a solution in which LiCl replaced NaCl but in which NaHCO3 and NaH2PO4 were at the same concentration as in Tyrode solution. Figure 7 shows that after perfusion with this solution for w hr the secretory response was greatly reduced whilst after 1 hr it was almost abolished. In this experiment the response of the gland to stimulation was also tested
SODIUM AND CATECHOLAMINE SECRETION 83 1 min after the sodium-deficient solution had begun to pass through the gland; if anything, the observed response was slightly less than the control response. In a similar experiment, normal responses to stimulation were obtained up to 12 min after the sodium-deficient solution had begun to pass through the gland. However, 1 min later the response was much reduced and after a further 55 min was almost abolished. DISCUSSION Inhibiting the sodium pump by treatment with ouabain or by omitting potassium ions from the extracellular fluid would be expected to result in a lowering of the intracellular concentration of potassium and a raising of the intracellular concentration of sodium. The present experiments, those of Banks (1967) and those of Douglas & Rubin (1961) show that perfusion with ouabain or with potassium-free media enhances both the basal secretion and that evoked by carbamyl- or acetylcholine. Furthermore, acetyl- and carbamylcholine are ineffective as secretagogues in Ca2+-free media and ouabain is without effect on the basal secretion under the same conditions (Douglas & Rubin, 1961; Banks, 1967). It would'appear, therefore, that the ability of calcium ions to act as the stimulus/secretion coupling agent is enhanced by either an elevated intracellular concentration of sodium or by a reduced intracellular concentration of potassium or by both of these factors. Perfusion with sodium-free solutions abolishes or severely reduces the secretory response both to injections of carbamylcholine and to raising the potassium concentration of the perfusing medium to 7 mm. As well as carbamylcholine, high concentrations of potassium are ineffectual as stimulating agents in the absence of Ca2+ ions (Douglas & Rubin, 1961). Thus the calcium-dependent secretion of catecholamines is abolished or greatly reduced by continued perfusion with sodium-free media. Such prolonged perfusions with sodium-free solutions might be expected to reduce the intracellular concentration of sodium to values lower than normal. Figure 7 shows that the secretory response is little affected immediately on changing to a sodium-deficient medium although the extracellular concentration of sodium must be very much lower than normal. Furthermore, normal responses can be obtained 12 min after perfusion with a sodium-deficient medium has begun and smaller, but nevertheless distinct, responses to stimulation can be demonstrated after half an hour. These observations indicate that secretory responses can be evoked at very low extracellular concentrations of sodium. The abolition or reduction of the secretory response in sodium-free media is probably, therefore, a consequence of lowering the intracellular concentration of
84 P. BANKS AND OTHERS sodium rather than of simply reducing the extracellular concentration of sodium. The finding that the secretory response is increased when the intracellular concentration of sodium is raised by perfusion with ouabain (Banks, 1967) or K+-free media whilst the extracellular concentration remains unchanged also suggests that the secretory response depends upon the intracellular concentration of sodium. If this view is correct, it is possible that sucrose maintains the secretory response in the absence of sodium better than Li or choline because it reduces the rate of sodium loss from the cells to a value lower than that occurring in LiCl or choline chloride based solutions. In the absence of external sodium, the spontaneous release of catecholamines at first rises above the control level and subsequently falls towards normal values. Such an effect might be anticipated if external sodium ions have an inhibitory action on calcium entry as has been described for squid and crab nerves (Baker et al. 1967a; Baker & Blaustein, 1968). However, the failure to demorstrate an increased response to stimulation immediately on changing to perfusion with a sodium-free medium would argue against this view. The experiments presented in this paper and that of Banks (1967) are consistent with the view that the Ca2+-dependent secretion of catecholamines is enhanced by elevated intracellular levels of sodium and is depressed by lowered levels. It is thus possible that calcium influx into chromaffin cells is regulated to some extent by the intracellular level of sodium as has been described for the axons of Loligo and Maia by Baker and his colleagues (1967a, b, 1968). The marked similarity between the results described above and those obtained by Milner & Hales (1968) for the secretion of insulin from slices of pancreas poses the question of whether a sodium-dependent Ca2+-influx is involved in the response of other types of secretory cell to stimulation. Indeed, since this work was completed two papers by Birks & Cohen (1968a, b) have appeared in which it is argued that the intracellular concentration of sodium regulates calcium entry and acetylcholine release at neuromuscular junctions. In a recent paper Sorimachi (1968) has claimed that the secretory response of the adrenal medulla of the rabbit is not diminished by replacing NaCl in the perfusing Locke solution with LiCl. However, his published figure shows that 25 min after changing to Li-Locke solution the response has fallen by about 3 %. Such an observation is in agreement with those described above. We are most grateful to Dr P. F. Baker for his encouragement and advice during the course of this work.
SODIUM AND CATECHOLAMINE SECRETION 85 REFERENCES BAKER, P. F. & BLALTSTEIN, M. P. (1968). Sodium-dependent uptake of calcium by crab nerve. Biochim. biophys. Acta 15, 167-17. BAKER, P. F., BLAUSTEIN, M. P., HODGKIN, A. L. & STEINHARDT, R. A. (1967a). The effect of sodium concentration on calcium movements in giant axons of Loligoforbesi. J. Physiol. 192, 43-44P. BAKER, P. F., BLAUSTEIN, M. P., MANIL, J. & STEINHARDT, R. A. (1967 b). A ouabainiinsensitive, calcium-sensitive sodium efflux from giant axons of Loligo. J. Physiol. 191, 1-12P. BANKS, P. (1966). The release of adenosine triphosphate catabolites during the secretion of catecholamines by bovine adrenal medulla. Biochem. J. 11, 536-541. BANKS, P. (1967). The effect of ouabain on the secretion of catecholamines and on the intracellular concentration of potassium. J. Physiol. 193, 631-637. BIR1KS, R. I. & COHEN, M. W. (1968a). The action of sodium pump inhibitors on neuromuscular transmission. Proc. R. Soc. B 17, 381-399. BIRKS, R. I. & COHEN, M. W. (1968b). The influence of internal sodium on the behaviour of motor nerve endings. Proc. R. Soc. B 17, 41-421. DOUGLAS, W. W. & RUBIN, R. P. (1961). The role of calcium in the secretory response of the adrenal medulla to acetylcholine. J. Physiol. 159, 4-57. EULER, U. S. V. & HAMBERG, U. (1949). Colorimetric determination of noradrenaline and adrenaline. Acta physiol. scand. 19, 74-84. MILNER, R. D. G. & HALES, C. N. (1967). The sodium pump and insulin secretion. Biochim. biophys. Acta 135, 375-377. MILNER, R. D. G. & HALES, C. N. (1968). The role of sodium and potassium in insulin secretion from rabbit pancreas. J. Physiol. 194, 725-743. SORIMACHI, M. (1968). Effects of alkali metals and other monovalent ions on the adrenomedullary secretion. Eur. Jnl Pharmac. 3, 235-241.