Subcellular localization of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalmus

Life Sciences, Vol. 31, pp. 1765-1768 Printed in the U.S.A.

Pergamon Press

SUBCELLULAR LOCALIZATION OF IMMUNOREACTIVE DYNORPHIN AND VASOPRESSIN IN RAT PITUITARY AND HYPOTHALAMUS C.J. Molineaux and B.M. Cox Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 (Received in final form June 14, 1982) Summary Dynorphin is found mainly in the particulate fraction of rat pituitary Rland and hypothalamus homogenates. Dvnorphin-like immunoreactivity (DYN-LI) from neurointermediate lobe (NIL) homoRenates migrates at the same rate as vasopressin-like immunoreactivity (AVP-LI), in sucrose density gradients, whereas DYN-LI from the hypothalamus appears to migrate principally in a less dense region of the gradient. This suggests that dynorphin and vasopressin from pituitary are present in organelles of similar size and density, while the bulk of the dynorphin in the hvpothalamus appears to be stored in a different subcellular organelle. Anterior lobe (AL) dvnorphin appears to migrate in two separate bands on density ~radients: the less dense band (slower) migrates at a similar rate to that of dynorphin and vasopressin from NIL. When ~-neo-endorphin was measured in sucrose gradients of NIL and hypothalamus, it was found to co-migrate with DYN-LI. The potent opioid peptide dynorphin has been shown to be present in the pituitary and hypothalamus of rats (i). The function of this neuropeptide in the pituitary is unknown, and little is known of its subcellular distribution. The finding that dynorDhin is present in the hypothalamo-hypophysial tract within vasoDressin-containing cell bodies (2) has led to speculation as to its function in the posterior pituitary. This communication describes the subcellular localization of dynorphin in pituitary and hypothalamus. Methods Male Sprague Dawley rats (250-300 g) were used in all studies. Rats were decapitated and the brains were removed immediately. The pituitary was lifted from the pituitary fossa, separated into anterior and neurointermediate lobes and placed on ice. Groups of i-i0 glands were homogenized in 2.0 ml of ice-cold 0.32 M sucrose (i0 mM Tris, pH 7.4). Hvpothalami were dissected according to the method of Glowinski and Iversen (3) and these were homogenized in 3 volumes of sucrose-tris. These homogenates were centrifuged at 1000g for i0 min. The pellets were re-homogenized in the same medium and were again centrifuged at 1000g for i0 min. Supernatants were pooled. In some experiments, the supernatant was centrifuged at 30,O00g for 30 min to yield a crude synaptosomal pellet. Continuous sucrose ~radients (0.6-1.6 M in lOmM Tris, pH 7.4) were prepared using a syringe-type Beckman gradient maker in 5/8 x 4" polyallomer tubes. 0.5 ml of the lO00g supernatant was layered over the top of the Eradient and each was centrifuged at 100,000g for various lengths of time. 0024-3205/82/161765-04503.00/0

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After centrifugation, 1.0 ml fractions were collected, and peptide content of each fraction was determined by radioimmunoassay after acidification of each fraction with acetic acid to a final concentration of 0.i M and heating at 95°C for i0 min. DYN-LI was measured using "Lucia" antiserum as described previously (i). AVP-LI was determined using antiserum purchased from Calbiochem, and ~-neoendorphin was estimated using antiserum described by Weber, et al., 1982 (4). Results When material from pituitary of hypothalamus was subjected to differential centrifugation, it was found that the majority of the DYN-LI was present in a particulate fraction which sedimented at 30,O00g (see Table I). Some loss of DYN-LI in NIL and hypothalamus is apparent. AVP-LI appears to be present in the same particulate fraction as DYN-LI, though a large amount of AVP-LI is found in the supernatant after 30,000g centrifugation (data not shown). TABLE I Subcellular Fractions of Pituitary and Hypothalamus: of Dynorphin Immunoreactivity

Fraction

NIL

Localization

Dynorphin-like Immunoreactivity AL Hypothalamus

Homogenate

2.26 ± 0.25

1.01 ± 0.084

2.73 ± 0.16

lO00g Supernatant

1.41 ± 0.15

0.799 ± 0.062

1.89 ± 0.II

1000g Pellet

0.240 ± 0.02

0.0508 ± 0.01

0.154 + 0.013

30,000 Supernatant

0.202 ± 0.034

0.0918 ± 0.007

0.0848 ± 0.0038

30,000 Pellet

1.08

0.827

1.67

± 0.091

+ 0.076

± 0.12

Values expressed as nmoles DYN-LI/gland using dynorphin (1-13) antiserum, N=5, means ± sen. The organelles containing dynorphin and vasopressin were further characterized by fractionation of the 1000g supernatant on continuous sucrose density gradients. In NIL homogenates, DYN-LI co-migrated with AVP-LI (see Fig. i), while in the hypothalamus, the majority of the DYN-LI was found to migrate more slowly than AVP-LI. AVP-LI, on the other hand, has a similar profile in these two tissues. A small fraction of the hypothalamic DYN-LI migrated in a similar position to the AVP-LI, forming a small shoulder on the main DYN-LI peak. In both tissues the distribution of ~-neo-endorphin-like immunoreactivitv (~-neo-end-LI) in the gradients corresponds to the distribution of DYN-LI (data not shown). When AL lO00g supernatant was subjected to similar gradient centrifugation, DYN-LI was found to migrate as two separate bands (see Fig 2). The band of lower density can be seen to correspond in rate of sedimentation'with the DYN-LI-containing material from (NIL). When ~-neo-endorphin was measured in these fractions, it was also found to correspond to the DYN-LI peak with lower density (data not shown).

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Subcellular Localization of Dynorphin

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S U C R O S E G R A D I E N T S OF NIL A N D H Y P O T H A L A M I C D Y N O R P H I N A N D VASOPRESSIN

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Figure i. Hypothalamus and NIL dynorphin and vasopressin. A 1000g supernatant of hypothalamic or NIL homogenate was applied to a 0.6-1.6 sucrose density gradient, spun for 90 min at 100,000g. 0.i mi fractions were collected. Sucrose density decreases from left to right. Discussion The present study demonstrates that dynorphin is present in subcellular organelles of discrete size and density similar to the neurosecretory vessicles which contain vasopressin (5). Some loss of dynorphin during preparation of subcellular fractions was apparent, particularly in the NIL and hypothalamus. This might suggest that dynorphin-containing organelles are damaged during preparation, and that the DYN-LI liberated from these organelles is broken down by proteolysis. Some AVP-LI also appeared to be released during preparation of these fractions, appearing in the 30,000g supernatant. The appearance of DYN-LI in a band which corresponds in rate and position of equilibrium to that of AVP-LI indicates that these two neuropeptides are stored in organelles of similar size and density. Since dynorphin had been found in the vasopressin containing magnocellular neurons of the paraventricular nucleus, coexistence of dynorphin and AVP within the same granules is possible. DYN-LI is found primarily in a less dense portion of hypothalamic gradients, which would suggest that the majority of hypothalamic dynorphin is stored in organelles which are less dense than those of the NIL. Other studies suggest that most of the DYN-LI in hypothalamus is not associated with neurons of the hypothalamo-hypophysial tract (6). A small proportion of the hypothalamic organelles containing DYN-LI is present in the same position of the gradient as is AVP-LI. The close association between ~-neo-endorphin and dynorphin has been confirmed at the subcellular level in hypothalamus and NIL. ~-neo-endorphin has also been demonstrated in the AL, where it comigrates only with the less dense band of DYN-LI.

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Subcellular Localization of Dynorphin

Vol. 31, No.s 16 & 17, 1982

SUCROSE G R A D I E N T S OF AL AND NIL DYNORPHIN

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Figure 2. Anterior and Neurointermediate Lobe Dynorphin and Vasopressin. A 1000g supernatant of AL or NIL homogenate was applied to a 0.6-1.6 M sucrose density gradient, and spun for various times at 100,000g. Sucrose density decreases from left to right. Acknowledgement We thank Taufiek Alhadi for skilled technical assistance. References i. 2. 3. 4. 5. 6.

GOLDSTEIN, A. and CHAZAROSSIAN, V.E., Proc. Nat. Acad. Sci. USA 77, 6207-6210 (1980). WATSON, S.J., AKIL, H., FISCHLI, W., GOLDSTEIN, A., ZIMMERMAN, F. and VAN WIMERSMA GREIDANUS, T.B. Science 216, 85-87 (1982). GLOWINSKI, J. and IVERSEN, L., J. Neurochem. 13, 655-669 (1966). WEBER, E., EVANS, C.J. and BARCHAS, J.D., Nature, in press, (1982). WEINSTEIN, H., MALAMED, S. AND SACHS, H., Biochem. Biophys. Acta 50, 386-389 (1961). MOLINFAUX, C.J., FEUERSTEIN, G. and COX, B.M., in preparation (1982).