Brain Research, 65 (1974) 475-487 475 ©Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands SUBCELLULAR LOCALIZATION OF DOPAMINE fl-HYDROXYLASE AND ENDOGENOUS NOREPINEPHRINE IN THE RAT HYPOTHALAMUS JOSEPH T. COYLE AND MICHAEL J. KUHAR Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 and Depart- ment of Pharmacology and Experimental Therapeutics, Johns Hopkins School of Medicine, Baltimore, Md. 21205 (U.S.A.) (Accepted July 4th, 1973) SUMMARY The subcellular distribution of endogenous norepinephrine, dopamine /5- hydroxylase (EC 1.14.2.1) and endogenous inhibitors to dopamine fl-hydroxylase have been examined in the rat hypothalamus. Sixty percent of the recovered nore- pinephrine and activity of dopamine fl-hydroxylase sedimented in the crude mito- chondrial (P2) fraction; fractionation on a linear sucrose gradient revealed that the particulate-bound amine and enzyme were localized in the synaptosomal fractions. After osmotic shock of the P2 fraction the released storage vesicles were purified by differential centrifugation or centrifugation on a sucrose gradient; 40-70~ of the dopamine fl-hydroxylase activity released from the P~, fraction was localized in the fractions reported to contain storage vesicles. In contrast, less than 107o of P2- norepinephrine was found in these fractions. The fraction from the gradient that had the highest specific activity of dopamine fl-hydroxylase was shown by electron micros- copy to be markedly enriched with large, dense-core vesicles. Endogenous inhibitors to dopamine fl-hydroxylase were found in all subcellular fractions with only a 2.5-fold difference between the least and most inhibitory fractions. INTRODUCTION In peripheral sympathetic neurons, norepinephrine has been shown to be highly concentrated in vesicleslZ,l~, 25. These storage vesicles also contain the final enzyme in the synthesis pathway for norepinephrine, dopamine fl-hydroxylase (DBH; 1.14.2.1), in addition to chromagranins and ATPg,la,IL Although values vary depend- ing upon the source of the vesicles, at least 50 7O of the total DBH within the vesicles is firmly bound to the vesicular membranO 4. In addition to their role in protecting