Gloria Cho

A reduction in the concentration of immunoreactive corticotropin, malanotropin and lipotropin in the brain of the aging rat

We have previously shown that the hypothalamic concentration of immunoreactive alpha-melanotropin (alpha-MSHi) is markedly lower in the aging female rat than in the young rat. The current view is that alpha-MSH is derived from corticotropin (ACTH), and ACTH, in turn, is derived from a large molecular-weight precursor (pro-opiocortin); pro-opiocortin also serves as the precursor to beta- and gamma-lipotropin (LPH). To ascertain if the age-related reduction in the concentration of alpha-MSHi may be a result of a decline in the production of pro-opiocortin, we determined the content of immunoreactive ACTH (ACTHi), alpha-MSH (alpha-MSHi), gamma-LPH (gamma-LPHi), and protein, in 3 regions of the brain of young (4 months) and old (26-28 months) female rats: the medial basal hypothalamus (MBH, the region containing the perikarya of the ACTH/MSH/LPH neurons), the preoptic anterior hypothalamus (POA), and the thalamus (regions containing axons of these neurons). The concentration of ACTHi, alpha-MSHi (mol/mg protein), or gamma-LPHi (U/mg protein) in the MBH of old rats was 30-50% of that in the MBH of young rats. Moreover, the concentration of ACTHi, alpha-MSHi or gamma-LPHi in the POA and thalamus of old rats was also lower than that in the POA and thalamus of young rats. Based on these findings, we propose that aging causes a reduction in the production of pro-opiocortin in the brain of the female rat and that such a change may be related to the altered function of the brain of the aged.

Aging-related reduced release of LH-releasing hormone from hypothalamic granules

The effects of aging on the secretory function of hypothalamic granules containing luteinizing hormone releasing hormone (LHRH) were examined, using chelated copper as a test substance. Granules prepared from old and young female rats were each incubated with various concentrations of CuATP and the kinetic constants of LHRH release estimated. The LHRH content of the hypothalamus of old female rats (3.4 +/- 0.1 ng; mean +/- S.E.; n = 3 determinations) was lower than that of young female rats (4.7 +/- 0.1 ng; n = 3). CuATP-stimulated LHRH release (pg/hypothalamus) from granules isolated from old animals was significantly (p less than 0.001) lower than that from granules isolated from young animals; Vmax being 58.5 +/- 1.6 pg (n = 36) and 76.7 +/- 2.7 pg (n = 34) of LHRH released/min, respectively. In contrast, the fractional amount of CuATP-stimulated release of LHRH from granules isolated from old and young rats was similar; Vmax being 60 +/- 0.5% and 62 +/- 1% of granule LHRH released, respectively. In addition, the apparent Km for CuATP-stimulated release of LHRH from granules of old and young rats was also similar, 5.1 and 5.5 microM copper, respectively. These results are suggestive that aging does not alter the responsiveness of LHRH-containing granules to a secretion-stimulus such as copper but it does alter the capacity (amount) of peptide released.

α‐MELANOCYTE‐STIMULATING HORMONE: A POSSIBLE NEURONAL MARKER OF THE AGEING BRAIN

Abstract— The amount of α‐melanocyte‐stimulating hormone (α‐MSH) in the entire hypothalamus as well as the amount of α‐MSH in free granule and synaptosome fractions of hypothalamic homogenates was investigated throughout the lifespan of female rats (1‐24 months). A 900 g supernatant fluid was prepared from hypothalami following homogenization in an iso‐osmotic sucrose solution, and free granules and synaptosomes containing α‐MSH were fractionated by means of continuous sucrose density gradient centrifugation. α‐MSH was quantified by radioimmunoassay. The total amount of α‐MSH in the hypothalamus, as well as the amount in free granules and synaptosomes prepared from hypothalami increased progressively from the 1st to the 5th month of life, and this increase was more pronounced in the free granules than in the synaptosomes. On the other hand, the amount of α‐MSH in the hypothalamus and the amount present in free granules and synaptosomes prepared from 5‐24‐month‐old animals decreased with age, and this decrease appeared to proceed at similar rates in both subcellular compartments. Based on these results, it is suggested that ageing of α‐MSH neurons in the hypothalamus is accompanied by a degeneration of the axons and/or an alteration in the biosynthetic and degradative activities of the neuron.

Castration of Male Rats Reduces the Capacity of Granules Isolated from the Median Eminence to Secrete Luteinizing Hormone-Releasing Hormone in Response to Copper

The effect of castration of male rats on the secretory function of median eminence area (MEA) granules containing luteinizing hormone-releasing hormone (LHRH) was examined, using copper as a test substance. LHRH granules, isolated from MEA of sham or castrated rats 1, 2, or 12 weeks postoperatively, were incubated with various concentrations of copper complexed to histidine (CuHis) and the kinetic constants of LHRH release estimated. CuHis-stimulated release of LHRH was found to be a saturable function of the concentration of CuHis. Castration did not alter the apparent Km for CuHis-stimulated release of LHRH; the Km being 3 and 16.9 microM copper for granules obtained from 1-2 weeks and 12 weeks sham-operated rats, respectively, and 3.7-5.6 and 10.2 microM copper for the granules of castrated rats, respectively. Moreover, castration did not alter the fractional amount of LHRH released in response to CuHis. In contrast, castration markedly reduced the actual amount of LHRH released in response to CuHis; the Vmax being 1,933 and 2,942 pg LHRH released/6 min/MEA for 1-2 weeks and 12 weeks sham-operated rats, respectively, and 782 and 757 pg for the castrated rats, respectively. Similarly, the LHRH content of the MEA of castrated rats was lower than that of the shams. These results are suggestive that castration reduces the capacity of the MEA granules to release LHRH in response to a secretion stimulus such as copper and that this is due not to alterations in the biochemical parameters underlying the release process itself but to a reduced level of MEA LHRH available for release.

Apparent Co‐Sequestration of Immunoreactive Corticotropin, α‐Melanot opin, and γ‐Lipotropin in Hypothalamic Granules

Abstract: In the present study, the question of whether immunoreactive α‐melanotropin (α‐MSH1), corticotropin (ACTH1), and β‐melanotropin (β‐MSH1) are co‐sequestered in hypothalamic granules of adult male rats was addressed. When a 900 ×g supernatant fluid prepared from a hypothalamic homogenate was fractionated on continuous sucrose density gradients under non‐equilibrium Conditions, two populations of particles containing α‐MSH1, ACTH1, or β‐MSH1 Were observed. However, when fractionated under equilibrium conditions, the two populations of particles containing α‐MSH1 ACTH1, or β‐MSH1 were recovered as a single band. This sedimentation characteristic indicates that the particles containing a given peptide differ in size but are similar in density. In their sedimentation, the small particles containing α‐MSH1, ACTH1, and β‐MSH1 are indistinguishable from granules containing α‐MSH1, whereas the large particles containing α‐MSH1 (ACTH1, and β‐MSH1 are indistinguishable from synaptosomes containing α‐MSH1, β‐MSH1 had an apparent molecular weight (M.W.) of about 5,000, which is similar to that of γ‐lipotropin. ACTH1 was comprised of three species of molecules: big (M.W. ≥ 10,000), 5.7K (M.W. ≌ 5,700), and 4.5K (M.W. ≌ 4,500). Big ACTH was the predominant and 5.7K ACTH the minor component of ACTH1 present in granules as well as in synaptosomes. These results are suggestive that α‐MSH, ACTH and its precursors, and γ‐lipotropin are co‐sequestered in hypothalamic granules.

Molecular-weight profiles of immunoreactive corticotropin in the hypothalamus of the aging rat

Extracts of the medial basal hypothalamus (MBH), the preoptic anterior hypothalamus (POA), or the pituitary gland of young (4-month-old) and old (18-month-old) female rats were fractionated on columns of Sephadex G-75 superfine. Five forms of immunoreactive corticotropin (ACTHi) were found in the MBH or POA: greater than 40K, 30-40K, 20-30K, 5.7K, and 4.5K. In contrast, 4 forms of ACTHi were found in the pituitary gland: 30-40K, 20-30K, 5.7K, and 4.5K. Thus, hypothalamic tissue contains a large form of ACTH (greater than 40K ACTH) which is not present in the pituitary gland. We tentatively identified greater than 40K ACTH as a large form of pro-opiocortin, 30-40K ACTH as pro-opiocortin, 20-30K ACTH as ACTH biosynthetic intermediate, 5.7K ACTH as glycosylated ACTH 1-39, and 4.5K ACTH as ACTH 1-39. The content of ACTHi in the MBH and POA of old rats was lower than that of young rats. Nevertheless, regardless of the age of the animals, the fractional amount of 30-40K ACTHi was high in the MBH (a region that includes the presumed site of biosynthesis of pro-opiocortin) compared to that in the POA (a region that is distant to the site of biosynthesis of pro-opiocortin). Moreover, the reduced fractional amount of 30-40K ACTHi in the POA was associated with an increased fractional amount of greater than 40K, 20-30K, 5.7K, and 4.5K ACTHi. These findings are consistent with a precursor-product relationship between the 30-40K ACTHi and 20-30K ACTHi, 5.7K ACTHi, and 4.5K ACTHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for synergism between copper and prostaglandin E2 in stimulating the release of gonadotropin-releasing hormone from median eminence explants: Na+/Cl- requirements.

Copper (Cu) and PGE2 are known to stimulate LHRH release from explants of the median eminence area (MEA) by two mechanisms distinguishable by their Ca2+ dependence. Moreover, exposure to Cu and PGE2 results in an amplified release of LHRH which is partially Ca2+ dependent, thus, resembling the release process stimulated by PGE2 alone. We have shown that LHRH release stimulated by Cu alone is Na+/Cl- dependent. By defining the Na+/Cl- dependence of PGE2- and Cu/PGE2-stimulated release of LHRH, we wished to ascertain if there is synergism between Cu and PGE2 actions. MEA of adult male rats were incubated for 5 min with 150 microM Cu and then for 15 min with 10 microM PGE2 (Cu/PGE2). Controls were incubated with Cu or PGE2. LHRH release into the medium was evaluated by RIA. Substituting Cl- in the incubation buffer with the non-permeant anion, isethionate, did not alter PGE2 stimulation of LHRH release, but it drastically inhibited Cu/PGE2 stimulation of LHRH release, indicating that this process requires a permeant monovalent anion. PGE2 and Cu/PGE2 stimulation of LHRH release were both inhibited when Na+ was substituted with Li+, or when 0.5 mM ouabain was included in the Na+-containing buffer; neither 10 microM tetrodotoxin (TTX) nor 100 microM amiloride were inhibitory. To ascertain if Na+ is required for Cu uptake, we evaluated the uptake of 67Cu by MEA explants and found that neither ouabain nor Li+ inhibited uptake, indicating that the extracellular Na+ and the activity of Na+/K+ ATPase are required for the process of LHRH release.(ABSTRACT TRUNCATED AT 250 WORDS)