Immunoreactive β-endorphin in the plasma, pituitary and hypothalamus of young and old male rats

Neurobiology of Aging, Vol. 2, pp. 281-284, 1981.Printedin the U.S.A.

Immunoreactive/3-Endorphin in the Plasma, Pituitary and Hypothalamus of Young and Old Male Rats L. J. F O R M A N , 1 W . E. S O N N T A G , 2 D. A. V A N V U G T a A N D J. M E I T E S 4

D e p a r t m e n t o f Physiology, N e u r o e n d o c r i n e R e s e a r c h Laboratory Michigan State University, East Lansing, M I 48824 R e c e i v e d 7 O c t o b e r 1981 FORMAN, L. J., W. E. SONNTAG, D. A. VAN VUGT AND J. MEITES. lmrnunoreactive [3-endorphin in the plasma, pituital3' and hypothalamus of young and old male rats. NEUROBIOL. AGING 2(4) 281-284, 1981.--Immunoreactive beta-endorphin (IR-/~-ENDO) was measured in the plasma, pituitary, and hypothalamus of young (3-5 too.) and old (19-23 mo.) male Sprague-Dawley rats, using a specific radioimmunoassay. Plasma IR-/3-ENDO in old male rats (3.44±0.54 ng/ml) was more than three times higher than values observed in young male rats (1.00-+0.10 ng/ml). Pituitary content and concentration of IR-/3-ENDO also were significantly greater in the old (5.85+-.0.51 p.g/gland and 1.17-+0.10p.g/mg protein) than in the young (3.53-+0.29 /zg/gland and 0.78-+0.06 /zg/mg protein) male rats. The content of IR-/3-ENDO in the hypothalamus of old and young rats was nearly the same (43.45-+2.47 and 49.88-+6.35 ng/hypothalamus, respectively), whereas the concentration of IR-/3-ENDO in the hypothalamus of the old male rats (3.89-+0.25 ng/mg protein) was approximately 50% lower than that observed in the young male rats (7.80-+0.85 ng/mg protein). These changes in plasma, pituitary, and hypothalamic IR-/3-ENDO may contribute to the increase in prolactin and decrease in gonadotropins observed in old male rats, since/3-ENDO administration is known to produce these effects on prolactin and gonadotropin secretion. Immunoreactive/3-endorphin

Plasma

Pituitary

IN old male rats, prolactin (PRL) release is increased and gonadotropin (Gn) release is reduced [4,20], and these changes in anterior pituitary (AP) hormone secretion are associated with a decrease in hypothalamic catecholamines (dopamine and norepinephrine), and a rise in hypothalamic serotonin metabolism [21]. It is well established that a reduction in hypothalamic catecholamine activity results in enhanced PRL and reduced gonadotropin secretion, whereas increased hypothalamic serotonin activity usually produces elevated PRL and decreased gonadotropin secretion [16]. Endogenous opioid peptides (EOPs) also have been shown to increase PRL and decrease Gn release from the AP [15], and at the same time to reduce dopamine and increase serotonin turnover in the hypothalamus [7, 11, 25, 26]. Thus the EOPs may participate in the rise of PRL and reduction in gonadotropin secretion observed in old rats. Recent reports have indicated that hypothalamic [24] and pituitary [13] concentrations of the opioid peptide, met 5enkephalin are increased in old male rats. Inasmuch as /3-endorphin (/3-ENDO) is the most potent of the EOPs

Hypothalamus

Aging

[15,18], is found in both the hypothalamus [3, 10, 27] and intermediate lobe of the pituitary [3,17], and detectable amounts of/3-ENDO are released by the pituitary into the systemic circulation [I,17], it was of interest to determine whether any significant differences could be observed in plasma, pituitary, or hypothalamic levels of/3-ENDO in old as compared to young male rats.

METHOD

Animals Young (3-5 mo. of age) and old (19-23 mo. of age) Sprague Dawley rats were obtained from Harlan Industries (Indianapolis, IN). The old male rats were obtained as retired breeders at 8-10 mo. of age, and were maintained in our colony until 1%23 mo. of age. All animals received food (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO) and water ad lib, and were maintained at 22°C on a 14:10 light/dark cycle (lights on at 0500 hr).

rAided by NIH Post-doctoral fellowship, AG05208 from the National Institute on Aging. 2Aided by NIH Post-doctoral fellowship, AG05147 from the National Institute on Aging. aPresent address: Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, 630 W. 168th Street, New York, NY 10032. 4Aided by NIH research grants AG00416 from the National Institute on Aging, CA10771 from the National Cancer Institute, and AM04784 from the National Institute of Arthritis, Metabolism and Digestive Diseases. Published with the approval of the Michigan Agricultural Experiment Station as Journal Article No. 9883.

C o p y r i g h t © 1981 A N K H O I n t e r n a t i o n a l Inc.--0197-4580/81/040281-04503.00/0

282

FORMAN E'l A d

Experimental Procedures Plasma was obtained from young and old animals by decapitation between 1000 and 1200 hrs. Trunk blood was collected into heparinized 30× 105 mm plastic centrifuge tubes to which 150/~1 of 200 IU heparin (Sigma Chemical Co., St. Louis, MO) in sterile saline was added. The blood was centrifuged at 1000×G in a Sorvall RC-2B refrigerated centrifuge for ten rain at 4°C. The resulting plasma was removed and frozen at -20°C until assayed for IR-B-ENDO. Pituitaries were removed and placed in 12×75 mm polystyrene test tubes, to which 2 ml of phosphate buffer (0.02 M NaPO4, 0.15 M NaCI, 10 mM EDTA, 0.01% sodium azide, and 0.1% gel, pH 7.5) containing 1 mM N-ethylmaleimide, had been added. Pituitaries were then homogenized and frozen at -20°C. At the time of decapitation, the whole brain was removed and quickly frozen on dry ice. Each brain was then partially thawed and the hypothalamus grossly dissected. Cuts were made 3 mm anterior to the optic chiasm and rostral to the mammillary bodies. Sections were bordered laterally by the lateral hypothalamic sulci and dorsally by a cut at the top of the third ventricle. The sections included the organuum vascuiosum of the laminae terminalis, the preoptic area, the supraoptic nucleus, the suprachiasmatic nucleus, the anterior hypothalamic area, the median eminence, the infundibular stalk, the arcuate nucleus, the ventromedial nucleus and the dorsomedial nucleus. Tissues were homogenized in buffer containing 1 mM N-ethylmaleimide and stored in 12x75 mm polystyrene tubes at -20°C. Pituitary and hypothalamic protein contents were measured by the technique of Lowry et a! [14]. Radioimmunoassay (RIA) of ~-ENDO Immunoreactive fl-ENDO was measured using a double antibody RIA. The first antibody (generously donated by Dr. S. S. C. Yen, University of California, San Diego, CA) was generated by injecting rabbits with human/3-endorphin conjugated to bovine serum albumin [28]. This antiserum was determined by us to exhibit a 30% cross-reactivity with /3-1ipotropin (NIAMDD-BLPH-RP1). fl-endorphin (Boehringer-Mannheim Biochemicals, Indianapolis, IN) was iodinated by modification of the chloramine-T method of Hunter and Greenwood [l 2]. Tracer binding was inhibited in a dose related manner with unlabeled/3-endorphin from 50 pg to 4000 pg. The minimal detectable dose of IR-/3-ENDO was 30 pg/tube with 50% inhibition of tracer binding, achieved with 500 pg/tube of unlabeled/3-endorphin. Parallelism to the standard curve was demonstrated by using increasing volumes of plasma, pituitaries, and hypothalami (Fig. 1). All determinations were made in duplicate. The intra- and interassay coefficients of variation were 7 and 9%, respectively. Statistical Analysis Statistical analysis of the data were performed using Student's t analysis. A significance level ofp<0,05 was chosen. RESULTS

A comparison of the values of IR-fl-ENDO in the plasma, pituitary, and hypothalamus of young and old male rats is shown in Table 1. Values for plasma and pituitary IR-/3ENDO represent the combined data from two separate determinations. In the old male rats. plasma values of IR-/3-

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ng/tube FIG. 1. Parallelism of various sample volumes of plasma (circles), pituitaries (squares) and hypothalami (triangles) from young.(open symbols) and old (closed symbols) male rats, to the 13-endorphin standard curve (stars).

ENDO were more than three times greater (3.44_+0.54 ng/ml) than those observed in young male rats (l.00±0.10 ng/ml; p<0.01). In addition, both the content and concentration of IR-fl-ENDO in the pituitaries of the old male rats (5.85 ±0.51 /zg/gland and 1.17--.0.10/xg/mg protein, respectively) were significantly greater (p<0.0l) than observed in the young male rats (3.53±0.29 gg/gland and 0.78___0.06 tzg/mg protein, respectively). No significant differences were observed in the hypothalamic content of IR-/3-ENDO between young (49.88±6.35 ng/hypothalamus) and old (43.45±2.47 ng/hypothalamus) male rats. However, when the data were expressed in terms of the concentration of IR-/3-ENDO in the hypothalamus, IR-fl-ENDO was observed to be reduced by approximately 50% in the old (3.89±0.25 ng/mg protein) as compared to that in the young (7.80±0.85 ng/mg protein) male rats (p<0.Ol), The protein content of the hypothalamus of young and old rats was 5.96__.0.14 mg/hypothalamus and 11.44__.0163 mg/hypothalamus, respectively, and was significantly greater (p<0.05) in the old male rats (Table 2). The suitability of our assay to measure IR43-ENDO in the rat is demonstrated in Fig. 1. Increasing sample volumes of plasma, pituitaries and hypothalami, respectively, caused the displacement of tracer from the first antibody equivalent to that of unlabeled/3-endorphin. In addition, column chromatography, using 1.5 x 60 cm glass columns of Bio-Gel P-60 revealed that rat plasma and pituitaries showed an immunoreactive peak which co-migrated with h-/3-endorphin (kindly supplied by Dr. C. H. Li, Hormone Research Labs, University of California, San Francisco, CA, data not shown). DISCUSSION

The present study revealed more than a three-fold greater

f l - E N D O R P H I N IN Y O U N G A N D O L D RATS

283

TABLE 1 IR-B-ENDORPHIN IN THE PLASMA, PITUITARY, AND HYI~THALAMUS OF YOUNG AND OLD MALE RATS

Plasma (ng/ml) Pituitary content (/.Lg/gland) concentration (~g/mg protein) Hypothalamus content (ng/hypothalamus) concentration (ng/mg protein)

N

Young*

N

Old*

22

1.00 ___0.1

22

3.44 ± 0.54t

22 22

3.53 -4- 0.29 0.78 ___0.06

22 22

5.85 +- 0.51t 1.17 _ 0.10t

12 12

49.88 ± 6.35 7,80 -x-_0.85

12 12

43.45 _ 2.47 3.89 ± 0.25t

*Each value represents a mean +_ one standard error. tp<0.01 when compared to young rats. N =number of animals per group.

TABLE 2 TOTAL PROTEIN CONTENT OF THE PITUITARYAND HYPOTHALAMUS OF YOUNG AND OLD MALE RATS

Young Old

N

Pituitary* (rag/gland)

Hypothalamus* (mg/hypothalamus)

22 12

4.15 ± 0.24 7.06 _ 1.34

5.96 -+ 0.14 11.44 ___0.63"~

*Each value represents a mean z one standard error. tp<0.01 when compared to young rats. N=number of animals per group.

concentration of plasma IR-/3-ENDO in old as compared to that present in young male rats. Pituitary content and concentration of IR-/3-ENDO also were significantly greater in the old than in young male rats, suggesting increased synthesis as well as release of pituitary B-ENDO in old male rats. The content of IR-/3-ENDO in the hypothalamus was about the same in old and young rats, but the concentration was 50% less in old than in young male rats. Since the content of IR-/3-ENDO is extremely low in the hypothalamus as compared to that present in the pituitary [3], we believe that most if not all of the plasma IR-/3-ENDO came from the pituitary. It has not been shown that hypothalamic B-ENDO is released into the systemic circulation. The physiological significance of the large increase in plasma IR-/3-ENDO in old male rats is not entirely clear at present. It is possible that the increase in plasma IR-/3E N D O partially contributes to the increase in plasma PRL and to the decrease in plasma Gn observed in old male rats [4,20], since systemic administration of/3-ENDO to rats elevates plasma PRL [5,18] and reduces plasma Gn concentrations [5,6]. Conversely, administration of the specific opiate antagonist, naloxone, decreases plasma PRL and elevates plasma Gn levels in rats [5]. The increase of IR-/3-ENDO in the plasma of old rats is not believed to contribute to the decrease in pulsatile GH secretion reported in old male rats

[23], since B-ENDO increases G H release [18]. We recently have presented evidence suggesting that the decrease in pulsatile GH secretion in old male rats probably is due to the reduction in hypothalamic catecholamine turnover [22]. It is possible that the increase in plasma IR-/3-ENDO in old male rats partially contributes to the decrease in hypothalamic dopamine and increase in hypothalamic serotonin metabolism found in old rats [21], since/3-ENDO has been reported to reduce hypothalamic dopahaine and to increase hypothalamic serotonin activity in rats [7, 11, 25, 26]. An alternative explanation for the increased release of/3-ENDO in the plasma of old male rats comes from recent work indicating that serotonin stimulates [19] whereas dopamine inhibits [9] IR-/3-ENDO release from the pituitary of rats. It is possible, therefore, that the reduction in hypothalamic dopamine and elevation of hypothalamic serotonin metabolism in old male rats [21] is not due to the EOPs but to other causes, and that these changes in hypothalamic amines are responsible for the increased release of IR-/3-ENDO into the plasma of old male rats. Our results on pituitary and hypothalamic concentrations or IR-/3-ENDO are in disagreement with those reported by Gambert et al. [8], who found pituitary and hypothalamic concentrations of IR-/3-ENDO to be unchanged with age in male rats. However, the data of Gambert et al. [8] show a high degree of variability, and in addition, did not include the more important assay of/3-ENDO in the plasma. It is well established that measurements of hormones in glands and tissues do not necessarily reflect release rates into the blood. In the present study, a 50% decrease in the concentration of IR-/3-ENDO in the hypothalamus was found. This appears to be in agreement with the data of Barden et al. [2] who also reported a 50 % reduction in hypothalamic concentration of IR-fl-ENDO in discrete hypothalamic nuclei of old as compared to young male rats. The decrease in hypothalamic IR-/3-ENDO concentration may indicate increased metabolism of this peptide in the hypothalamus, which could contribute to the increase in blood PRL and decrease in blood Gn levels in old male rats.

284

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