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We have previously described the presence and wide distribution of a luteinizing hormone (LH)-like peptide, widely distributed in the rat central nervous system, with highest levels in the hypothalamus. We have found that intracerebroventricular injection of 100 micrograms colchicine causes a significant rise in hypothalamic LH, from 549 +/- 170 pg/mg protein (n = 12) in controls to 1,679 +/- 279 pg/mg protein (n = 13) in treated animals, p less than 0.01. There was no associated change in levels of LH in the pituitary. Since colchicine stops axoplasmic flow, these findings of colchicine-induced increase in hypothalamic LH indicate that at least a portion of hypothalamic LH is present in long-axoned neuronal elements with cell bodies within the hypothalamus and axons extending to the extrahypothalamic brain. Thus, hypothalamic LH does not solely represent measurement of LH in cells of the contignous pars tuberalis.

Our laboratory has previously described the widespread distribution of an immunoreactive and bioactive rat growth hormone (rGH)-like protein in rat brain. It has also been demonstrated that regulation of pituitary rGH secretion is at least partly mediated by a short-loop negative feedback system. In such a system, increased levels of rGH, acting at a suprapituitary locus, would decrease pituitary GH secretion. Thus, the present study has dealt with attempts to further investigate the hypothesis that one function of brain-based rGH might be as a mediator of the short-loop negative feedback system controlling pituitary rGH release. If brain-based rGH were to function as a mediator of such a system, then in situations where serum rGH levels are decreased, brain rGH concentrations should increase, indicating activation of a negative feedback loop. In the present communication we report that significantly decreased serum GH levels in oophorectomized and in thyroidectomized rats were coupled with a significant increase in rGH concentrations in the hypothalamus and in the amygdala. By contrast, adrenalectomy, which was not associated with any changes in levels of GH in serum caused no perturbations in levels of rGH in the brain. These discordant changes in serum and brain-based rGH are findings compatible with the hypothesis that one function of brain-based rGH is as a mediator of the short-loop negative feedback system regulating the release of pituitary GH.

Ultracentrifugation and sucrose gradient separation techniques have been applied to study of the subcellular distribution of hypothalamic luteinizing hormone (LH). Subcellular particle-bound LH accounted for 42% of the total LH while cytoplasm contained 58% of the total immunoassayable LH in these extracts. 70% of particle-bound LH was present in the P2 fraction and most of the particle-associated LH was found in subfractions of P2 rich in synaptosomes but poor in myelin and mitochondria. While actual amounts of LH in the P3 and P4, the microsomal fractions, were relatively small, the concentration of LH, expressed as nanograms per milligram of protein was quite high. Both content and concentration of LH in the nuclear pellet, P1, were substantially lower. These findings provide further framework for speculation on the role of brain-based LH.

Pursuant to our identification of prolactin-like immunoreactivity (PLI), widely distributed in rat brain, the spinal cord was examined for the presence of this pituitary-hormone-like protein. PLI was present in all spinal cord extracts examined and averaged 500 +/- 53 pg/mg protein. Hypophysectomy, causing a fall in serum prolactin to undetectable levels, was not associated with any change in levels of PLI in spinal cord. Recovery of rat prolactin standards added to spinal cord homogenates was 97.6 +/- 3.9%. When increasing concentrations of spinal cord extract were assayed in a prolactin radioimmunoassay, displacement of rat 125I-Prolactin from antiserum was parallel to that displacement produced by increasing concentrations of rat anterior pituitary standards. Upon subjection to gel permeation chromatography, the elution profiles of immunoreactive prolactin from spinal cord were different from the profiles of anterior pituitary prolactin. In addition to an immunoreactive prolactin peak eluting with pituitary prolactin, spinal cord extracts showed a large void volume peak and late eluting low-molecular-weight materials not seen with anterior pituitary. In the Nb2 lymphoma cell assay, all spinal cord extracts demonstrated prolactin-like bioactivity with a bioactivity/immunoreactivity ratio of 1.05 +/- 0.13. We conclude: (1) PLI, widely distributed in rat brain, is also present in spinal cord; (2) spinal cord prolactin levels are independent of levels in pituitary and peripheral circulation; (3) this immunoreactive prolactin is bioactive, and (4) differing gel permeation chromatographic elution profiles indicate that there may be some molecular differences between pituitary and spinal cord prolactin.

Characterization of the molecular, immunological and biological properties of a thyrotropin-stimulating hormone (TSH)-like peptide in rodent brain tissue showed similarities to its pituitary counterpart. The distribution of immunoreactivity in rodent brain was determined by radioimmunoassay in both intact and hypophysectomized animals. Easily detectable but smaller quantities of immunoassayable TSH were present in formalin fixed, acetone-stored brains from Macaca mullata. No change in the level of this TSH-like peptide was observed in most rat brain parts, although the hypothalamus did show a significant drop in hormone level after removal of the pituitary. Extracts of brain containing TSH-like material restored thyroid histology to normal when administered to hypophysectomized rats. Tissue cultured neural cells from both intact and hypophysectomized rats released a TSH-like material into the medium over a 30-day time period. Sodium l-thyroxine suppressed TSH release from pituitary monolayers but did not alter TSH release from brain derived cells that were similarly cultured. Surgical thyroidectomy resulted in a striking rise in serum TSH concentrations with no alteration in the content of brain TSH.

We have recently reported immunoassayable luteinizing hormone (LH) in several areas of the rat brain and conspicuously present in the hypothalamus. In this report, we focus on the presence of LH in the hypothalamus and its potential role in regulation of pituitary LH release. In adult female rats, examined during the course of the estrous cycle, a significant fall in hypothalamic LH coincides with the surge in pituitary and serum LH at the time of proestrus, signaling ovulation. Ovariectomized adult rats show no change in hypothalamic LH at a time when there is a dramatic rise in both anterior pituitary and serum LH. These data support the concept that hypothalamic LH is not of pituitary origin and that it may play a role in the short-loop negative feedback system controlling the surge in anterior pituitary LH release, and event initiating ovulation.

Prompted by immunohistochemical reports of prolactin-like immunoreactivity in cell bodies within the rat hypothalamus, a study was undertaken to quantitate the immunologic and biologic activity of this material. Hypothalamic concentrations of prolactin-like immunoreactivity averaged 402 +/- 23 pg/mg of protein (n = 30). 97% recovery of rat prolactin standards added to homogenates of hypothalamus insured that neuronal tissue, as prepared for these studies, did not interfere with the radioimmunoassay of rat prolactin. Examination of the elution profile from Sephadex G-75 columns of the prolactin-like immunoreactivity in hypothalamic extracts showed that the majority of hypothalamic prolactin-like substance was of a larger molecular size than pituitary prolactin. While increasing amounts of brain extract progressively displaced more I125 prolactin from antibody-binding sites, the displacement curve produced by adding hypothalamic extract was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Hypothalamic extracts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoactivity (B/I) ratios for hypothalamic extracts were significantly lower than the B/I ratios for pituitary prolactin (0.71 +/- 0.04 for pituitary, vs. 0.19 +/- 0.06 in the hypothalamus; p less than 0.001). Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in the hypothalamus, indicating that brain prolactin-like substance is regulated independently of pituitary prolactin and circulating serum prolactin levels.

We have recently reported that luteinizing hormone (LH) is present in the hypothalamus of rats. It has chromatographic and biologic characteristics similar to pituitary LH. In this report we focus on extrahypothalamic LH that is widely distributed in the rodent central nervous system. This material has a chromatographic profile similar to that of pituitary LH. Serial dilution of this material is parallel with dilutions of rat pituitary LH in the immunoassay. Brain extracts are active in the testis LH radioligand receptor assay and in the rat interstitial cell testosterone secretion bioassay. Prior incubation of extract with LH antibody significantly attenuated both of these activities. Thus, extrahypothalamic LH has immunologic, chromatographic, and biologic characteristics similar to hypothalamic and pituitary LH.