Molecular Pharmacology, Vol 9, 105-116, Copyright © 1973 by the American Society for Pharmacology and Experimental Therapeutics

Recovery of Rat Adrenal Amine Stores after Insulin Administration

¹ Department of Physiology and Pharmacology and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Acute adrenal medullary discharge of catecholamines was produced in response to the hypoglycemia caused by a large dose of insulin (5 IU/kg, intravenously). Rats were killed 4, 24, 48, 72, or 96 hr after drug administration. Intact adrenal storage vesicles were separated from empty vesicles by differential and density gradient centrifugation, and all fractions were analyzed for catecholamines, dopamine -hydroxylase (a marker for vesicle membranes), and ATP. Four hours after insulin, vesicular catecholamines and ATP fell to 25% of control levels, while vesicular dopamine -hydroxylase fell to 40%; the enzyme activity increased in the broken vesicle membrane fraction. The ability of isolated storage vesicles to incorporate [¹⁴C]epinephrine fell to about 25% of controls, while incorporation of [³H]metaraminol fell only to 65%. Twenty-four hours after insulin administration vesicular ATP, dopamine -hydroxylase, and [¹⁴C]epinephrine incorporation returned to 50-60% of controls, while [³H]metaraminol incorporation returned to normal; vesicular catecholamine content remained at about 25%. Catecholamine recovery consistently lagged behind all other parameters; by 96 hr vesicular catecholamines, ATP, and [¹⁴C]epinephrine incorporation approached control levels, while vesicular dopamine -hydroxylase exceeded control values. Isopycnic sucrose density gradient centrifugation of vesicles from control rats, and from rats given insulin 24 hr previously, indicated that the newly synthesized storage vesicles were able to incorporate both isotopically labeled epinephrine and metaraminol, but that the new vesicles had a lower equilibrium density than the original population of vesicles. They also had a lower catecholamine content and a lower specificity for epinephrine relative to metaraminol than in the controls. The efflux of endogenous or newly incorporated epinephrine from the new vesicles was nearly identical with that of the controls. These data suggest that the sequence of recovery of adrenal amine stores after sympathetic discharge proceeds by the following process: (a) resynthesis of vesicles (reappearance of vesicular dopamine -hydroxylase) with restoration of ability to incorporate metaraminol; (b) restoration of storage mechanism (ATP) and epinephrine incorporation; (c) restoration of catecholamines; (d) oversynthesis of new vesicles (as determined by dopamine -hydroxylase measurements).

Note:
ACKNOWLEDGMENT The authors wish to thank Mrs. Nannie Jordan for her technical assistance.

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