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Metabolic requirements for secretion from the adrenal medulla
Life Sciences Vol. 8, Part 1, pp . 799-803, 1989 . Printed in Great Britain
Pergamon Press
METABOLIC REQUIREMENTS FOR SECRETION FROM THE ADRENAL MEDULLA+ N . Kirshner and W. J . Smith Departments of Biochemistry and Experimental Surgery Duke University Medical Center Durham, North Carolina (Received 26 March 1989 ; in final form 30 April 1989) The effects of metabolic inhibitors on secretion from the adrenal medulla were investigated to obtain further information about the secretary process . Previous studies have shown; (a) , that stimulation-secretion coupling requires Ca m (1) ; (b) , that acetylcholine stimulated the uptake of Cam by the medulla cells (2) ; (c) , that during secretion adenine nucleotides (3) and a specific protein (4, 5) contained within the storage vesicles were released ; (d) , that stimulated secretion did not require oxygen (6) ; and (e) , that the secretary process had an apparent Qi° of 2 to 3 (7) . Methods Bovine adrenal glands were obtained 20 to 30 minutes after slaughter . The glands were cannulated through the adrenal vein, perfused with 20 to 30 ml of ice cold Locke's solution (154 mM NaCl, 5 .6 mM KCl, 2 .2 mM CaClZ, 2 .15 mM Na2HP0,~, 0 .86 mM NaH2P0,,, 10 mM glucose) and transported in ice to the laboratory . The glands were then perfused with Locke's solution at 30° for 30 to 40 minutes at a flow rate of 4 ml,/ min before samples for assay were collected . Stimulation was evoked by perfusing for 2 minutes with Locke's solution containing acetylcholine (10 -s q/ ml) . Samples were collected at i-minute intervals, centrifuged to remove any particulate matter, diluted with distilled water and assayed, without prior purification on alumina, for adrenallno and 799
800
ADRENAL MEDULLA SECRETION
Vol . 8, No . 15
noradrenaline by the trihydroxyindole method (8) . When the effects on secretion of cyanide and iodoacetate were tested, these compounds were present in the Locke's solution at a concentration of 1 mM . The glands were periused with these solutions for 10 minutes prior to the collection of samples, and throughout the collection period . Acetylcholine was added to the Locke's solution containing cyanide, or iodoacetate or both . Results The results obtained are shown in Figs . 1 and 2 . In Fig . 1 the response of the gland to stimulation with acetylcholine in normal Locke's solution is shown at A. After perfusiog for 10 minutes with KCN-Locke's solution, the response is shown at B and again after a 10-minute perfusion with IAA-Locke's solurtion at C . When both cyanide and IAA were present in the perfusion medium, the response to acetylcholine was completely blocked (D) . The decreased response at B (Fig . 1) may be due to a partial block by cyanide . However, in these experiments, and in those reported by Douglas (9) . there was always a decreased response to repetitive stimulation although perhaps not quite as marked as in Fig . 1 . The data of Fig . 1 and 2 indicates that the contribution of cyanide to the blockage of secretion is reversible whereas the effect of IAA is irreversible . When the gland was periused with IAA-Locke's solution (Fig . 1C) following perfusion with KCN-Locke's solution (Fig . 1B) there was no further inhibition . However, when the gland was perfused with KCN-Locke's solution (Fig . 2B) following perfusion with lAA-Locke's solution (Fig . 2A) secretion was completely blocked . When the gland was then perfused with normal Locke's solution, the response to acetylcholine was restored and could again be blocked ?, y perfusiog with KCN-Locke's solution and again restored by perfusioq with
Vol . 8, No . 15
ADRENAL MEDULLA SECRETION
KCN
IAA
801
IAAtKCN
FIG . 1 Effect of cyanide and iodoacetate on secretion from the adrenal medulla Glands were perfused as described in the text . Each of the bars represents a 2-minute collection period . The solid horizontal bars indicate the time acetylcholine was present in the perfusion medium . Between each part of the experiment, the gland was perfused for 10 minutes with Locke's solution containing 1 mM KCN at B, 1 mM IAA at C and 1 mM IAA and 1 mM KCN at D. normal Locke's solution . The data in Figs . 1 and 2 indicate that a metabolic source of energy is required to evoke secretion and that either glycolysis or oxidative phosphorylation can fulfill this requirement . ATP or phosphoenolpyruvate added to the perfusion medium did not overcome the blockage due to IAA plus cyanide . Whether this energy requirement is for one or more discrete steps in the secretory process itself ci whether it is required for maintaining the structural and
802
ADRENAL MEDULLA SECRETION
Vol . 8, No . 15
functional integrity of the medulla cells is not known .
FIG . 2 Effect of cyanide and iodoacetate on secretion from the adrenal medulla Each of the bars represents a 2-minute collection period . The solid horizontal bars indicate the time acetylcholine was present in the perfusion medium . Ten minutes bèfote collecting samples at A the gland was perfused with Locke's solution containing 1 m~I IAA . Between the subsequent parts of the experiment, the glad was perfused for 10 minutes with Locke's solution containing 1 mM KCN at B and D and with normal Locke's solution at C and E . Summary Secretion of catecholamines evoked by acetylcholine from isolated perfused bovine adrenal glands was completely inhibited when cyanide and iodoacetate were both present in the perfusion medium at a concentration of 1 mM . Neither cyanide nor iodoacetate by themselves prevented secretion . However,
.'ol. 8, No. 15
ADRENAL MEDULLA SECRETION
803
when the gland was first perfused with iodoacetate, then cyanide did block secretion . The contribution of cyanide to the inhibitory effect was reversible but that of iodoacetate was not . These studies indicate that a metabolic source of energy is required either for the secretory process itself or to maintain the cell in a functional state . Acknowledgements This work was supported by a grant from the National Institutes of Health (AM-05427) . A report of this work was presented at the Spring, 1966 meeting of the National Academy of Sciences, Science, 154, 422 (1966) . #Career Development Awardee, National Institutes of Health (K3-GM-15, 184) . ##
Advanced Research Fellow, American Heart Association . Present address: Biology Department, East Carolina College, Greenville, N .C . References
1.
Douglas, W. W. and Rubin, R . P ., J . Physiol ., 15~, 40 (1961) .
2.
Douglas, W. W. and Poisner, A . M ., J. Physibl ., 162, 385 (1962) .
3.
Douglas, W. W. and Poisner, A. M ., J . Physiol ., 183, 249 (1966) .
4.
Banks, P . and Helle, K ., Biochem . J ., L7, 40C (1965) .
5.
Kirshner, N ., Sage, H . J ., Smith, W. J ., and Kirshner, A. G ., Fed . Proc 25, 735 (1966) .
6.
Banks, P ., Biochem . J., Q7, 555 (1966) .
7.
Kirshner, N . , Sage, H . J . and Smith, W. J. , Mol . Pharmacol . , (1967) .
8.
Einer, U . S . von, and Lishajko, F ., Acta Phvsiol . 9cand ., 51 . 348 (1961)
9.
Douglas, W. W. and Rubin, R . P ., 1 . Phvsiol ., 167. 288 (1963) .
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Pergamon Press
METABOLIC REQUIREMENTS FOR SECRETION FROM THE ADRENAL MEDULLA+ N . Kirshner and W. J . Smith Departments of Biochemistry and Experimental Surgery Duke University Medical Center Durham, North Carolina (Received 26 March 1989 ; in final form 30 April 1989) The effects of metabolic inhibitors on secretion from the adrenal medulla were investigated to obtain further information about the secretary process . Previous studies have shown; (a) , that stimulation-secretion coupling requires Ca m (1) ; (b) , that acetylcholine stimulated the uptake of Cam by the medulla cells (2) ; (c) , that during secretion adenine nucleotides (3) and a specific protein (4, 5) contained within the storage vesicles were released ; (d) , that stimulated secretion did not require oxygen (6) ; and (e) , that the secretary process had an apparent Qi° of 2 to 3 (7) . Methods Bovine adrenal glands were obtained 20 to 30 minutes after slaughter . The glands were cannulated through the adrenal vein, perfused with 20 to 30 ml of ice cold Locke's solution (154 mM NaCl, 5 .6 mM KCl, 2 .2 mM CaClZ, 2 .15 mM Na2HP0,~, 0 .86 mM NaH2P0,,, 10 mM glucose) and transported in ice to the laboratory . The glands were then perfused with Locke's solution at 30° for 30 to 40 minutes at a flow rate of 4 ml,/ min before samples for assay were collected . Stimulation was evoked by perfusing for 2 minutes with Locke's solution containing acetylcholine (10 -s q/ ml) . Samples were collected at i-minute intervals, centrifuged to remove any particulate matter, diluted with distilled water and assayed, without prior purification on alumina, for adrenallno and 799
800
ADRENAL MEDULLA SECRETION
Vol . 8, No . 15
noradrenaline by the trihydroxyindole method (8) . When the effects on secretion of cyanide and iodoacetate were tested, these compounds were present in the Locke's solution at a concentration of 1 mM . The glands were periused with these solutions for 10 minutes prior to the collection of samples, and throughout the collection period . Acetylcholine was added to the Locke's solution containing cyanide, or iodoacetate or both . Results The results obtained are shown in Figs . 1 and 2 . In Fig . 1 the response of the gland to stimulation with acetylcholine in normal Locke's solution is shown at A. After perfusiog for 10 minutes with KCN-Locke's solution, the response is shown at B and again after a 10-minute perfusion with IAA-Locke's solurtion at C . When both cyanide and IAA were present in the perfusion medium, the response to acetylcholine was completely blocked (D) . The decreased response at B (Fig . 1) may be due to a partial block by cyanide . However, in these experiments, and in those reported by Douglas (9) . there was always a decreased response to repetitive stimulation although perhaps not quite as marked as in Fig . 1 . The data of Fig . 1 and 2 indicates that the contribution of cyanide to the blockage of secretion is reversible whereas the effect of IAA is irreversible . When the gland was periused with IAA-Locke's solution (Fig . 1C) following perfusion with KCN-Locke's solution (Fig . 1B) there was no further inhibition . However, when the gland was perfused with KCN-Locke's solution (Fig . 2B) following perfusion with lAA-Locke's solution (Fig . 2A) secretion was completely blocked . When the gland was then perfused with normal Locke's solution, the response to acetylcholine was restored and could again be blocked ?, y perfusiog with KCN-Locke's solution and again restored by perfusioq with
Vol . 8, No . 15
ADRENAL MEDULLA SECRETION
KCN
IAA
801
IAAtKCN
FIG . 1 Effect of cyanide and iodoacetate on secretion from the adrenal medulla Glands were perfused as described in the text . Each of the bars represents a 2-minute collection period . The solid horizontal bars indicate the time acetylcholine was present in the perfusion medium . Between each part of the experiment, the gland was perfused for 10 minutes with Locke's solution containing 1 mM KCN at B, 1 mM IAA at C and 1 mM IAA and 1 mM KCN at D. normal Locke's solution . The data in Figs . 1 and 2 indicate that a metabolic source of energy is required to evoke secretion and that either glycolysis or oxidative phosphorylation can fulfill this requirement . ATP or phosphoenolpyruvate added to the perfusion medium did not overcome the blockage due to IAA plus cyanide . Whether this energy requirement is for one or more discrete steps in the secretory process itself ci whether it is required for maintaining the structural and
802
ADRENAL MEDULLA SECRETION
Vol . 8, No . 15
functional integrity of the medulla cells is not known .
FIG . 2 Effect of cyanide and iodoacetate on secretion from the adrenal medulla Each of the bars represents a 2-minute collection period . The solid horizontal bars indicate the time acetylcholine was present in the perfusion medium . Ten minutes bèfote collecting samples at A the gland was perfused with Locke's solution containing 1 m~I IAA . Between the subsequent parts of the experiment, the glad was perfused for 10 minutes with Locke's solution containing 1 mM KCN at B and D and with normal Locke's solution at C and E . Summary Secretion of catecholamines evoked by acetylcholine from isolated perfused bovine adrenal glands was completely inhibited when cyanide and iodoacetate were both present in the perfusion medium at a concentration of 1 mM . Neither cyanide nor iodoacetate by themselves prevented secretion . However,
.'ol. 8, No. 15
ADRENAL MEDULLA SECRETION
803
when the gland was first perfused with iodoacetate, then cyanide did block secretion . The contribution of cyanide to the inhibitory effect was reversible but that of iodoacetate was not . These studies indicate that a metabolic source of energy is required either for the secretory process itself or to maintain the cell in a functional state . Acknowledgements This work was supported by a grant from the National Institutes of Health (AM-05427) . A report of this work was presented at the Spring, 1966 meeting of the National Academy of Sciences, Science, 154, 422 (1966) . #Career Development Awardee, National Institutes of Health (K3-GM-15, 184) . ##
Advanced Research Fellow, American Heart Association . Present address: Biology Department, East Carolina College, Greenville, N .C . References
1.
Douglas, W. W. and Rubin, R . P ., J . Physiol ., 15~, 40 (1961) .
2.
Douglas, W. W. and Poisner, A . M ., J. Physibl ., 162, 385 (1962) .
3.
Douglas, W. W. and Poisner, A. M ., J . Physiol ., 183, 249 (1966) .
4.
Banks, P . and Helle, K ., Biochem . J ., L7, 40C (1965) .
5.
Kirshner, N ., Sage, H . J ., Smith, W. J ., and Kirshner, A. G ., Fed . Proc 25, 735 (1966) .
6.
Banks, P ., Biochem . J., Q7, 555 (1966) .
7.
Kirshner, N . , Sage, H . J . and Smith, W. J. , Mol . Pharmacol . , (1967) .
8.
Einer, U . S . von, and Lishajko, F ., Acta Phvsiol . 9cand ., 51 . 348 (1961)
9.
Douglas, W. W. and Rubin, R . P ., 1 . Phvsiol ., 167. 288 (1963) .
3 2 54