Glucose-deprivation and nitrogen, when imposed either separately or

Transcription

1 J. Phyaiol. (1969), 202, pp With 2 text-ftgurem Printed in Great Britain THE METABOLIC REQUIREMENTS FOR CATECHOLAMINE RELEASE FROM THE ADRENAL MEDULLA BY R. P. RUBIN From the Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, U.S.A. (Received 25 November 1968) SUMMARY 1. The metabolic requirements for catecholamine secretion elicited by acetylcholine or by calcium plus high K+ were studied on acutely denervated perfused cat adrenal glands. 2. Glucose-deprivation plus anoxia caused an increase in the spontaneous catecholamine output from adrenal glands perfused with normal Locke solution, which was abolished by the removal of calcium from the perfusion medium. 3. Anoxia plus glucose-deprivation did not depress the secretary response to repeated exposures of a low concentration of acetylcholine, but did depress the response to a higher concentration of acetylcholine. Glucose-deprivation and nitrogen, when imposed either separately or together, did not inhibit total catecholamine output in response to calcium. Differential analysis of the calcium-evoked secretion showed that during anoxia, catecholamine output was maintained primarily by adrenaline secretion. 4. Cyanide (0x2 mm) potentiated thy secretary response to calcium in the presence of glucose, but when glucose was omitted from the perfusion medium, cyanide caused a gradual decline in calcium-evoked secretion. Iodoacetic acid (IAA) (0-2 mm) depressed the response to calcium by about 50% under aerobic conditions and by 90 % under anaerobic conditions. 5. The glycogen content of medullae was profoundly depleted under anoxic conditions. 6. It is concluded that energy is required for the secretary action of calcium on medullary chromaffin cells. The energy may be derived from glycolysis or oxidative metabolism. A possible interaction between calcium and adenosine triphosphate acid (ATP) in eliciting catecholamine secretion is discussed. 7. The alteration in the percent adrenaline and noradrenaline secreted

2 198 R. P. RUBIN during anoxia indicates that anoxia may regulate medullary catecholamine secretion through a peripheral, as well as a central mechanism. INTRODUCTION Previous work has established the absolute requirement for calcium in the mechanism of secretion of a variety of hormones and neurohumoral substances from a number of secretary organs including the adrenal medulla (Dougglas & Rubin, 1961, 1963; for references see Douglas, 1968). However, the exact role played by calcium in the secretary mechanism remains to be elucidated. ATP, but not calcium, is able to enhance the release of catecholamines from isolated chromaffin granules in vitro (Poisner & Trifaro, 1967) and it has been proposed by these workers that the action of calcium in vivo requires the presence of high energy phosphate intermediates. The experiments presented in this paper examine the metabolic requirements for calcium and for the acetylcholine-evoked catecholamine release from the perfused cat adrenal gland in order to determine whether the action of calcium in eliciting secretion does indeed directly depend upon the normal energy metabolism of the chromaffin cell. METHODS Adrenal perfusion. Cat adrenal glands acutely denervated were perfused in aitu at room temperature according to the method of Douglas & Rubin (1961). The perfusion medium was either normal Locke solution which had the following composition (mm): NaCl 154; KCl 5-6; CaCl2 2; MgCl3 0-2; NaHCO3 12; dextrose 10; or modified Locke solution containing excess K (56 mm), with the sodium chloride reduced by an equivalent amount to maintain isotonicity. In the solutions containing high potassium, calcium chloride was added when needed to give a final concentration of either 0 5 or 3-0 mm. Some solutions were equilibrated with 95 % oxygen and 5 % carbon dioxide; others were bubbled with 95 % nitrogen and 5 % carbon dioxide (see Results). All solutions had a ph close to 7 0. Catecholamine analysis. Samples of perfusate, which were collected by means of a cannula tied into the adrenolumbar vein, were acidified and subsequently analysed for catecholamines by a modification (Rubin & Jaanus, 1966) of the photofluorometric method of Anton & Sayre (1962). Outputs were expressed as total catecholamine (adrenaline plus noradrenaline) jug base/min. Glycogen determination. Glycogen analysis was carried out on the medulla of both perfused and non-perfused adrenal glands. The glands were removed from the animals as quickly as possible. The medulla was then separated from cortex, weighed, and placed in 2 ml. potassium hydroxide (30 mg/100 ml.) Some glands were kept for 24 hr in absolute ethanol at 5 C before the determinations were carried out. Storing the tissue under these conditions does not affect the glycogen content (Dixit & Lazarow, 1967). The glycogen content was then determined according to the general method of Seifter, Dayton, Novic & Muntwyler (1950). However, two modifications were made in order to increase the sensitivity of the method. Only 2 ml. anthrone reagent was used, and samples were read at 620 min, using a multiple sample absorbance recorder (Gilford 2000). When known amounts of

3 ENERGY METABOLISM AND MEDULLARY SECRETION 199 glycogen were carried through the chemical procedure, % recovery was obtained in the concentration range of 5-100,g, and a linear relationship was obtained between the amount of glycogen and the optical density. Values were expressed as,tg glycogen/mg medulla. In certain experiments, glycogen determinations were carried out on individual glands; however, in other experiments, where indicated, the analyses were done on pooled glands from two animals. It was established in four experiments that the mean glycogen content of the left adrenal medulla ( ,ug/mg) did not differ from that of the right medulla ( ). Thus, the percentage of glycogen depletion of the left (perfused) gland was calculated by relating the glycogen concentration in the perfused gland as a percent of the glycogen concentration of the non-perfused right gland, according to the following formula: Percentage glycogen depletion = (1 glycogen concentration of left gland \ glycogen concentration of right gland Agents used. Acetylcholine chloride (Nutritional Biochemical); sodium cyanide (Merck); iodoacetic acid (Nutritional Biochemical); and anthrone (Mann). RESULTS The effect of anoxia and glucose deprivation on catecholamine secretion Spontaneous catecholamine release. The spontaneous secretion of medullary catecholamines remains at a fairly constant level when the adrenal gland is perfused with normal Locke solution (Table 1). The removal of TABLE 1. Effect of anoxia and glucose deprivation on spontaneous catecholamine release Mean catecholamine output (,tg/min) ± s.e. Perfusion No. of, A medium expts. Control 0-5 min* 5-15 min* min* min* O2+glucose glucose-free N2+glucose N2+glucose-free * Time interval after perfusion was switched from normal Locke solution to media lacking glucose and/or oxygen. Control values in normal Locke solution were obtained from 2 min samples obtained just before the introduction of the test media. glucose from the perfusion medium did not augment the rate of secretion over a 30 min period (Table 1). However, when the perfusion solution was bubbled with a nitrogen-carbon dioxide mixture rather than with oxygencarbon dioxide, a very small increase in the resting secretion was seen (Table 1). When anaerobic conditions were employed, in the absence of exogenous glucose, there was a more pronounced and consistent increase in the spontaneous secretion of catecholamines over the 30 min period studied (Table 1). Although there was some variation from gland to gland, the peak rate of secretion was observed during the min following the introduction of the anoxic and glucose-deprived medium. The mean catecholamine output during this period ( ,tg/min) represented

4 200 R. P. RUBIN a tenfold increase in the rate of release, as compared to that observed in normal Locke solution ( ,ug/min). The spontaneous discharge, both during perfusion with normal Locke solution and following the introduction of glucose-lack plus nitrogen, was overwhelmingly in the form of adrenaline. The resting secretion, before anoxia and glucose-lack as percent adrenaline was o/ (mean of four experiments + s.e.). During the 30 min of perfusion with the N2 containing glucose-free medium, the mean percent adrenaline was 91x5 + 2x3 %. The increase in the spontaneous release of catecholamines during anoxia plus glucose deprivation was depressed by the removal of calcium from the perfusion medium. Samples collected during the min of perfusion with a calcium-free medium showed a mean output of 0x19 + 0x06,tg/min (five glands), compared to 0x98 4ag/min in the presence of calcium. The rate of spontaneous catecholamine secretion during anoxia and glucose deprivation was, therefore, depressed by more than 80% when calcium was absent from the perfusion medium. The enhanced resting secretion observed during anoxia plus glucose deprivation was not abolished by concentrations of hexamethonium (104 M) plus atropine (5 x 106 M) which depress completely the secretary response to maximal splanchnic nerve stimulation. Thus, when a single adrenal gland was perfused with Locke solution containing hexamethonium plus atropine the control rate of secretion was 0412 /g/min. The rate of spontaneous release increased to 0*97,g/min during the 25-30th min of perfusion with the nitrogen-containing glucosefree medium in the continued presence of hexamethonium plus atropine. Following prolonged periods of perfusion (1 hr) in the anaerobic and glucose-deprived medium, the rate of spontaneous release fell to lower levels than those observed during the first 30 min. The reintroduction of oxygen plus glucose to the perfusion medium caused a rapid augmentation of the secretion of catecholamines over the 5 min period after the normal medium was restored. Thus, in three glands studied, restoration of the normal medium increased the mean catecholamine output from to /tg/min. Acetylcholine-evoked secretion. Despite the stimulant effect of anoxia and glucose deprivation on spontaneous catecholamine secretion in normal Locke solution, some experiments were carried out to investigate the metabolic requirements for secretion elicited by high and low doses of acetylcholine. The secretary response to repeated exposures to acetylcholine (10 6g/ml.) was well maintained when either the glucose was absent (Fig. 1 c) or when nitrogen replaced oxygen in the perfusion medium (Fig. 1 d). The restoration of either glucose (Fig. 1 c) or oxygen (Fig. 1 d) to the Locke solution did not alter the response to this low concentration of acetylcholine. Even anoxia plus glucose deprivation together did not

5 ENERGY METABOLISM AND MEDULLARY SECRETION 201 diminish the enhanced outputs obtained during repeated exposures to acetylcholine (Fig. lb). In fact, the secretary responses to acetylcholine were better maintained in the anaerobic, glucose-free medium than in normal Locke solution (Fig. la, b). In the absence of glucose and oxygen the spontaneous rates of release just before the addition of acetyl- - -ab ~~~~~~~~~~~~~~~~~ min min o oi G-lucse-fre-LoGe p I*lucose-free+Ne ly W Locke C ~~~~~c d bo 2 ~~~~~~~~~~~~~~~6 01n S min min 0 *Glucose-free --14*--* *-N2-O----N--* 2 Locke Locke Fig. 1. The effect of anoxia and glucose-deprivation on the secretary response to acetylcholine. Adrenal glands were perfused for 30 min with either normal Locke solution (Fig. l a), Locke solution lacking both glucose and oxygen (Fig. 1 b), or Locke solution lacking either glucose (Fig. 1 c), or oxygen (Fig. 1 d). Acetylcholine was then added for 2 min periods every 7 min; the fourth response to acetylcholine was obtained after restoration of the normal perfusion medium. The stippled vertical columns depict the outputs obtained during stimulation with acetylcholine, 10-6 g/ml., and the clear vertical columns depict the evoked outputs obtained with 10-5 g/ml. acetylcholine. The scales on the left and right hand of the Figure refer to the catecholamine outputs obtained with low and high concentrations of acetylcholine, respectively. The results illustrated were obtained from individual experiments, with a different preparation used for each set of four responses to a given concentration of acetylcholine.

6 202 B. P. BUBIN choline ranged from 0 11 to ag/min. When 02 plus glucose were reintroduced to the perfusion medium, the secretary response to acetylcholine was enhanced (Fig. 1 b). When a tenfold higher concentration of acetylcholine (10-5 g/ml.) was employed to elicit secretion, the glucose-deprived, nitrogen-containing medium was not able to maintain the output during repeated exposures to this secretogogue to the same extent as did normal Locke solution (Fig. la, b). In fact, the third response to this concentration of acetylcholine, which normally elicits near maximal rates of secretion, was only 2-3 times higher than the resting rate of secretion. When oxygen and glucose were reintroduced into the medium, the response to acetylcholine was restored to some degree (Fig. lb). Glucose deprivation alone did not significantly depress the catecholamine output induced by the high concentration of acetylcholine (Fig. 1 c). The response to acetylcholine did appear to diminish somewhat more rapidly in the presence of nitrogen (Fig. ld), although not as precipitously as when glucose was also lacking (Fig. 1 b). The return of oxygen to the perfusion medium did not enhance the response to acetylcholine, but, in fact, appeared to inhibit it even further (Fig. 1 d). Calcium-evoked secretion. Since the spontaneous release of catecholamines was not significantly augmented during anoxia when perfusion was carried out in the absence of calcium, it was decided to study in greater detail the metabolic requirements for evoked-secretion by reintroducing calcium after periods of perfusion with a calcium-free high potassium (K+) medium. Neither the absence of glucose nor the presence of nitrogen depressed the secretary activity of the lower and higher concentrations of calcium (Fig. 2a, b, c). In fact, in the presence of nitrogen plus glucose the activity of calcium as a secretogogue was enhanced. The potentiating effect of the nitrogen was reduced by the removal of glucose from the perfusion medium (Fig. 2a, b, c). When both oxygen and glucose were restored to the perfusion medium for 5-7 min and stimulation with calcium (3.0 mm) repeated, the mean output increased to ,ag/min (three experiments), compared to the mean output of x2 and jug/min obtained previously with 3 0 mm calcium in the glucose-free plus nitrogen medium. The differential secretion of adrenaline and noradrenaline was profoundly altered under anoxic conditions. Under control conditions calcium elicited catecholamine outputs which were % noradrenaline (Fig. 2). However, in the presence of nitrogen, the percent noradrenaline fell to % (Fig. 2). The decrease in the percentage noradrenaline secreted during anoxia was the result of an augmentation in the discharge ofadrenaline together with a decrease in the secretion ofnoradrenaline. For example, 44 % of the 4-6,/g/min catecholamine secretion, elicited by 0*5 mm

7 ENERGY METABOLISM AND MED ULLARY SECRETION _ (5) a 8 (5) (5) (6) 4 4 b (3) -~~~~~~18 ~~~~~~~(4) -0 O12 (4) 15 _ (4)~~~~~~~~~~~~~~~~~4 6 T c (4) 10 ; t ;11 C)~~~~ I.L 4 ( (3) (4) 4 Fig.2. CONT. GL-free N2 N2 CN CN IAA!AA Fg2.The effect L-free L-fre N2 of inhibition of metabolism on calcium-evoked catecholamine secretion. Glands were perfused with calcium-free, high K+ Locke solution; 0-5 mm calcium was then added during the 30-32nd min and 3 0 mm calcium was added during the 37-39th min and 44-46th min of perfusion. The ability of calcium to elicit secretion was tested according to this general procedure, under control conditions (CONT.) and with the following modifications of the perfusion solution: glucose-free (GL-free); nitrogen (N2); nitrogen plus glucose-free (N2+ GL-free); cyanide 2 x 10-4 M (CN); cyanide plus glucose-free (CN + GL-free); iodoacetate 2 x 10-4 M (IAA); iodoacetate plus nitrogen (IAA + N2). The vertical columns in Fig. 2a depict the mean total catecholamine outputs (± s.e.) obtained with 0-5 mm calcium, and the columns in Fig. 2b and c depict total outputs obtained with the two responses to 3-0 mm calcium. The crosshatched and clear portion of each column represent the noradrenaline and adrenaline outputs, respectively. A different preparation was used for each set of three responses. The numbers in parentheses indicate the number of glands employed for each determination

8 204 R. P. RUBIN calcium, was noradrenaline and 56 % was adrenaline, which represents 2*0 /ag/min noradrenaline and 2 6,ug/min adrenaline. In the presence of nitrogen, 0 5 mm calcium evoked a mean output of 7 0,ug/min catecholamine and only 20 % was noradrenaline (Fig. 2a). Thus, under anoxic conditions calcium elicited 1x4 Itg/min noradrenaline and 5-6 /tg/min adrenaline. In response to 3-0 mm calcium, under aerobic and anaerobic conditions the adrenaline secretion was 6-2 and 11 6 /ug/min respectively, whereas the noradrenaline secretion was 3-5 and 1-8 Itg/min, respectively. Even when the normal medium was restored, following exposure to nitrogen plus glucose deprivation, and the secretary response to calcium augmented, the percentage noradrenaline was only % (three experiments). Thus, the enhanced output was the result of an increase in adrenaline secretion. The effect of metabolic inhibitors on calcium-evoked catecholamine secretion Cyanide. The addition of sodium cyanide (CN) (0.2 mm) to the calciumfree high K+ perfusion medium consistently produced an increased response to low and high concentrations of calcium (Fig. 2a, b, c). The enhancement of secretion by CN was of a greater magnitude than that caused by nitrogen, and the secretion, like that seen with nitrogen, was mainly in the form of adrenaline (Fig. 2). In the presence of CN, calcium-evoked secretion was only 11-16% noradrenaline. The removal of glucose from the perfusion medium progressively diminished the potentiating effect of CN on calcium-evoked secretion. Thus, the two exposures to calcium (3.0 mm) in the CN and glucose-free medium elicited outputs which were considerably below those obtained with calcium in high K+ (Fig. 2b, c). Iodoacetate. In the presence of IAA (0-2 mm) the response of the first exposure to calcium (0.5 mm) was depressed by about 50 % (Fig. 2a). When the same concentration of IAA was added in the presence of nitrogen, the initial response to calcium (0.5 mm) was reduced by almost 90 % (Fig. 2 a) and the depressed secretion could not be enhanced under these conditions by the higher concentration of calcium. Thus, in the presence of JAA the mean output in response to 3 0 mm calcium was ,ug/min (four experiments), as compared to ,ug/min when 0 5 mm calcium was the stimulating agent. The effect of anoxia and glucose-deprivation on the glycogen content of the adrenal medulla An unstimulated adrenal gland, which was perfused with normal Locke solution, manifested no diminution in glycogen content (Table 2). Sequential exposures to both a low and a high concentration of acetylcholine also

9 ENERGY METABOLISM AND MEDULLARY SECRETION 205 produced no marked depletion of glycogen. Excess K+ plus calcium caused approximately a 35 % decrease in the glycogen content of the medulla. Glucose deprivation alone did not further augment the glycogen depletion produced by either acetylcholine or excess K+ plus calcium; however, perfusion with a medium equilibrated with nitrogen (with or without glucose deprivation) did produce a % decrease in the glycogen content of perfused adrenal medullae exposed to stimulation by acetylcholine or calcium plus excess K+. The addition of CN alone to the perfusion medium did not appear to enhance glycogen depletion significantly; however, the glycogen content was depressed by about 65 % when glucose deprivation was imposed together with the CN (Table 2). TABLE 2. Expt. Locke (unstimulated) ACh (10-6) ACh (10-5) Ca2+ Ca2+ ACh (10-5) + glucose-freet Ca2 + glucose-freet ACh (10-6) + N2 + glucose-free ACh (10-5) + N2 + glucose-freet ACh(10-5)+N2t Ca2+ + N2 Ca2+ + N2 Ca2+ + NaCN Ca2+ + NaCNt Ca2+ + NaCN + glucose-freet Ca2+ + NaCN + glucose-freet The effect of anoxia, cyanide, and glucose-deprivation on the glycogen content of adrenal medullae Glycogen (Itg/mg tissue) Left gland* Right gland (perfused) (unperfused) * Stimulation of perfused glands with ACh and Ca2+ carried out in manner described in Figs. 1 and 2. t Glycogen determinations carried out on pooled glands from two cats. Percentage depletion DISCUSSION The present experiments have shown that the secretary activity of calcium on the medullary chromaffin cells was not significantly inhibited by the withdrawal of either glucose or oxygen from the perfusion medium. Indeed, even when deprivation of both glucose and oxygen were applied simultaneously, calcium was repeatedly able to elicit high rates of catecholamine secretion. This indicates that either the action of calcium to elicit secretion does not require the normal function of the metabolic processes of the chromaffin cell, or that the chromaffin cell is still able to supply needed energy despite anoxia and glucose deprivation

10 206 R. P. RUBIN The finding that anoxia and glucose deprivation did gradually depress the secretary response to repeated exposures to a high concentration of acetylcholine suggests that during periods of high activity the substrate reserves of the cell might eventually be exhausted. Glycogen can serve as an energy source in the absence of oxygen and exogenous substrate. The cat adrenal medulla contains a significant amount of glycogen, which is profoundly reduced during anoxia. Thus, even during the conditions of anoxia and glucose deprivation, energy would be temporarily produced by anaerobic glycolysis, utilizing the glycogen as a substrate; however, the eventual depletion of this substrate would lead to insufficient energy production and a suppression of evoked secretion. The block of oxidative metabolism by CN did not reduce, but, in fact, enhanced the secretary response. However, when the effects of CN were combined with glucose deprivation, repeated exposures to calcium eventually depressed the ability of the chromaffin cells to secrete. Under these experimental conditions, the glycogen stores were also severely depleted. In order to prevent the utilization of glycogen as a source of energy production, IAA was employed, since in 0-2 mm concentration it selectively blocks glycolysis by inhibiting the enzyme, 3-phosphoglyceraldehyde dehydrogenase (see Webb, 1966). Indeed, IAA plus anoxia, which should depress both glycolysis and oxidative phosphorylation, produced almost complete inhibition of the secretary action of calcium. These results indicate that simultaneous inhibition of both the glycolytic and oxidative pathways is required to block completely the secretary action of calcium. The present data are strikingly similar to those obtained from studies done some time ago on sympathetic ganglion cells, which are developmental homologues of the medullary chromaffin cells. Larrabee & Bronk (1952) concluded that ganglionic transmission could be maintained for some time under anaerobic conditions, even in the absence of exogenous glucose, by an acceleration of glycolysis, utilizing intracellular substrate reserves such as glycogen. The present data are also in general agreement with previous studies indicating that interference with metabolism can depress the activity of secretary cells (for references, see Douglas, 1968). The question arises as to the mechanism by which the secretary action of calcium is intimately bound to the energy-producing mechanisms of the chromaffin cell. The interference with calcium entry into the cell might account for the effects observed. However, the inhibition of calciumelicited secretion was not reversed by higher concentrations of calcium. By contrast, local anaesthetics do interfere with calcium influx and efflux from perfused cat adrenal glands, and their inhibitory action on calciumevoked secretion can be readily reversed by excess calcium (Rubin, Feinstein, Jaanus & Paimre, 1967; Rubin & Miele, 1968a). Furthermore,

11 ENERGY METABOLISM AND MEDULLARY SECRETION 207 it seems more likely that the entry of calcium into the medullary chromaffin cell is a passive process rather than an active one involving a saturable carrier; for over the concentration range of 0 5 up to at least 17-6 mm, the evoked catecholamine output varies directly with the calcium concentration in the perfusion medium (Douglas & Rubin, 1961). It is deemed more likely that the interference with the normal metabolic functions of the chromaffin cell somehow uncouples the link between the entry of the calcium into the cell and the eventual extrusion of the catecholamine. Recent evidence has established that the chromaffin secretary granules are the source of the catecholamines released during in vivo stimulation (Douglas, Poisner & Rubin, 1965; Banks & Helle, 1965; Blaschko, Comline, Schneider, Silver & Smith, 1967; Kirshner, Sage & Smith, 1967). Since calcium does not significantly enhance the release of catecholamines from medullary granules in vitro (Hillarp, 1958; Banks, 1966; Poisner & Trifaro, 1967), it appears that the action of calcium to trigger secretion requires some intermediate step which involves the presence of energy stores. The finding that ATP has a dose-dependent stimulant effect on isolated medullary granules (Poisner & Trifaro, 1967), and the suggestion by these authors that this nucleotide might be the link which initiates secretion following the entry of calcium into the chromaffin cell, are both consistent with the present data. However, the nature of the possible interaction between calcium and ATP, as of the present time, must be conjectural. Although anoxic conditions, in the absence of exogenous glucose, did eventually produce a decrease in the total catecholamine output evoked by acetylcholine or calcium, an analysis of the differential secretion of adrenaline and noradrenaline during anoxia showed a striking alteration in discharge of these two hormones. The percent noradrenaline secreted decreased sharply so that during anaerobiosis catecholamine output was maintained by an augmentation in the discharge of adrenaline. Thus, noradrenaline-containing cells seem to be more strongly dependent on aerobic metabolism for their secretary activity than are adrenalinecontaining cells. It is well known that glycolysis can be accelerated by lack of oxygen, a phenomenon referred to as the Pasteur Effect. Apparently, during anoxia, glycolysis is accelerated in the adrenaline-storing cells, as evidenced by the increased adrenaline discharge both under anoxic conditions and after the subsequent restoration of the normal perfusion medium. Previous investigations have shown that the adrenaline- and the noradrenaline-containing chromaffin cells of the cat adrenal gland can be distinguished by their localization within the medulla (Rubin, Cohen, Harman & Roer, 1968), and by certain different pharmacologic properties (Jaanus, Miele & Rubin, 1967; Rubin & Miele, 1968b). The present data

12 208 R. P. RUBIN show that the adrenaline and noradrenaline cells also have biochemically distinguishable properties. The results of the present study have also shown that anoxia, when combined with glucose deprivation, can greatly augment the resting secretion of catecholamines by a direct action on the chromaffin cell. The catecholamine secreted consisted almost entirely of adrenaline and was dependent on the presence of calcium in the perfusion medium. Since anoxia is unable to enhance the secretion of catecholamines from isolated chromaffin granules (Blaschko, Hagen & Welch, 1955), it is presumed that the stimulant action of anoxia is related to some effect on the chromaffin cell membrane. The prompt enhancement of the rate of spontaneous catecholamine release when glucose and oxygen were restored to the perfusion medium supports the idea of an increase in the permeability of the chromaffin cell during anoxia and glucose deprivation. Furthermore, it has been well established that interference with the normal metabolic processes results in the depolarization of excitable tissue such as isolated nerve (see Brink, 1956). It is well known that anoxia can elicit large outputs of catecholamines in the whole animal by means of neurally controlled reflex pathways. However, asphyxia can also augment catecholamine secretion from denervated cat and dog adrenal glands perfused with blood (Vogt, 1952; Biilbring, Burn & DeElio, 1948); and in the foetal calf-adrenal gland, the response to asphyxia appears to be independent of its nerve supply (Comline & Silver, 1966). These results, as well as those of the present study, give support to the theory that the effects of anoxia on medullary chromaffin cells can be regulated through peripheral as well as central mechanisms. The skilful and enthusiastic assistance of Miss Eleanor Roer was extremely valuable in carrying out these experiments. Martin J. Rosenstein performed the glycogen determinations during a student research externship. This study was supported by research grant AM 09237, from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service. REFERENCES ANTON, A. H. & SAYRE, D. F. (1962). A study of the factors affecting the aluminium oxidetrihydroxyindole procedure for analysis of catecholamines. J. Pharmac. exp. Ther. 138, BANKS, P. (1966). An interaction between chromaffin granules and calcium ions. Biochem. J. 101, 18-20c. BANKS, P. & HELLE, K. (1965). The release of protein from the stimulated adrenal medulla. Biochem. J. 97, C. BLAsCHKO, H., HAGEN, P. & WELCH, A. (1955). Observations on the intracellular granules of the adrenal medulla. J. Phyiol. 129, BLAScKmo, H., COMLINE, R. S., SCHNEIDER, F. H., SILVER, M. & SMITH, A. D. (1967). Secretion of a chromaffin granule protein, chromogranin, from the adrenal gland after splanchnic stimulation. Nature, Lond. 215,

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