Early development of the thyroid axis in the Brattleboro rat.

Plasma concentrations of thyrotrophin (TSH), thyroxine (T4), and triiodothyronine (T3), and pituitary TSH concentrations were determined at weekly intervals during the first 42 days following birth in Brattleboro homozygous (DI), Brattleboro heterozygous (HZ), and Long-Evans (LE) rats. Offspring from matings of Brattleboro rats were divided into DI and HZ animal subgroups on the basis of hypothalamic vasopressin content. In control LE rats, circulating levels of TSH, T4, and T3, and pituitary TSH concentrations increased during the early postnatal period to reach relatively stable levels between 28 and 42 days of age. In DI and HZ rats, the thyroid axis developed in parallel to that of LE rats during initial postnatal weeks. However, by 42 days of age, pituitary TSH concentrations were clearly elevated in Brattleboro rats relative to levels in age-matched LE animals. These data indicate that differences in thyroid axis function between Brattleboro and LE rats occur only after the attainment of a degree of maturity.

Immunoreactive prolactin in the rat hypothalamus: in vitro release and subcellular localization.

Immunocytochemical studies have identified immunoreactive prolactin (IR-PRL) in the hypothalamus and other areas of the rat brain. However, neither the release of IR-PRL from the hypothalamus nor its subcellular localization have been demonstrated. In this study, the release of IR-PRL from hypothalami obtained from female rats was examined using hypothalamic units incubated in vitro in Krebs-Ringer bicarbonate-glucose buffer. Hypothalamic tissue spontaneously released IR-PRL, and this release was increased by depolarizing concentrations of potassium by a calcium-dependent mechanism. Hypothalamic IR-PRL was also released from hypothalamic tissue obtained from hypophysectomized rats (14 days). The subcellular localization of IR-PRL was investigated using equilibrium-density centrifugation. Tissue homogenates from intact or hypophysectomized rats were centrifuged at 150 g at 4 degrees C for 10 min, and the supernatants were layered onto continuous sucrose gradients (1.00-1.27 g/ml) and centrifuged at 100,000 g (max.) for 16 h. IR-PRL in pituitary supernatants showed a high equilibrium-density peak with a modal density of 1.23 g/ml. Fractionation of the supernatant from ventral or dorsal hypothalamic tissue resulted in two high-equilibrium density peaks, a primary peak with a modal density of 1.23 g/ml and a smaller peak with a modal density of 1.10 g/ml. Both high-density peaks were maintained in tissue obtained from hypophysectomized rats and were disrupted by homogenization in hypo-osmotic medium. Together, these data suggest that hypothalamic IR-PRL is stored in membrane-bound particles which have densities similar to those of secretory granules and is released by a calcium-dependent mechanism when the tissue is depolarized.

Subcellular localization of immunoreactive thyroid-stimulating hormone in the rat hypothalamus.

Hypothalamic tissue contains TSH-like material which is biologically, immunologically, and physicochemically similar to pituitary TSH. Immunoreactive thyroid-stimulating hormone (IR-TSH) is released from hypothalamic tissue in vitro by depolarizing concentrations of potassium or veratridine by a calcium-dependent mechanism. In the present study, we investigated the subcellular localization of IR-TSH using equilibrium density centrifugation. Tissue homogenates from intact, hypophysectomized or thyroidectomized rats were centrifuged at 150 g at 4 degrees C for 10 min and the supernatants were layered onto continuous sucrose gradients (1.00-1.27 g/ml) and centrifuged at 100,000 g (max) for 16 h. IR-TSH in pituitary supernatants from intact and thyroidectomized rats showed high equilibrium density peaks with a modal density around 1.2 g/ml. Fractionation of the supernatant from ventral or dorsal hypothalamic homogenates resulted in a bimodal distribution of IR-TSH. In supernatants from both tissues, IR-TSH containing particles were found at the top of the gradient in a low equilibrium density peak between 1.0 and 1.08 g/ml. In addition, IR-TSH containing particles were found in ventral and dorsal hypothalamic supernatants with modal densities at 1.16 and 1.25, respectively. These high density IR-TSH particles were present in tissue taken from hypophysectomized rats, and were not appreciably affected by thyroidectomy. Homogenization of the tissue in a hypo-osmotic medium disrupted the high density IR-TSH particles resulting in a single low density peak at the top of the gradient. These data suggest that hypothalamic IR-TSH is stored in membrane bound particles which have densities similar to that of secretory granules.

Distribution and release of immunoreactive thyroid-stimulating hormone in the rat hypothalamus: effects of thyroidectomy, hypophysectomy and treatment with thyroid hormones.

Immunoreactive thyroid-stimulating hormone (IR-TSH) has been detected in the hypothalamus and is released in vitro by a calcium-dependent mechanism when the tissue is depolarized. Recently, immunocytochemical studies have revealed that IR-TSH is present in thyrotropes in the pars tuberalis. Therefore, because these thyrotropes are associated with the median eminence, the area with the highest concentration of IR-TSH, it is of interest to determine if 'hypothalamic' IR-TSH is from neural or pituitary cells. We addressed this issue by studying the effects of hypophysectomy, thyroidectomy, or chronic administration of triiodothyronine (T3) or thyroxine (T4) on the distribution and in vitro release of IR-TSH in the hypothalamus. We reasoned that, if hypothalamic IR-TSH is dependent on the thyrotropes of the pars tuberalis, then changes in hypothalamic IR-TSH concentration and release should be parallel to those measured in pituitary extracts. IR-TSH was measured in tissue extracted in ice-cold 2% NaCl, with a final pH of 4.5. For the in vitro studies, tissues were incubated for 20-min periods in Krebs-Ringer bicarbonate buffer at 37 degrees C. In untreated rats, the concentration of IR-TSH is greater in the ventral than the dorsal portion of the hypothalamus (39.3 +/- 8.2 vs. 4.0 +/- 1.5 ng/mg wet wt.). Upon finer dissection of the hypothalamus into median eminence and anterior, middle, and posterior portions of the remainder, IR-TSH was only detectable in the middle hypothalamus (5.3 +/- 1.5 ng/mg), and the median eminence (149 +/- 41 ng/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Release of immunoreactive thyroid stimulating hormone from the rat hypothalamus in vitro.

Thyroid stimulating hormone (TSH) has been identified in the hypothalamus and other brain areas of the rat. However, the alteration of the release of brain TSH has not been demonstrated. Therefore, we examined the release of immunoreactive TSH (IR-TSH) in vitro from hypothalamic tissue obtained from hypophysectomized, thyroidectomized and intact control rats. Whereas TRH (10(-5) M) and PGE2 (10(-4) M) did not alter hypothalamic IR-TSH release, depolarizing concentrations of potassium (60 mM) or veratridine (5 mM) stimulated the release of IR-TSH from hypothalamic tissue from all groups. These data suggest that IR-TSH synthesized in the hypothalamus is stored in a releasable form.

Pharmacological studies of the involvement of hypothalamic prostaglandins in the regulation of thyrotropin secretion.

A case is made for the involvement of pituitary prostaglandins (PGs) in the regulation of thyrotropin (TSH) secretion by citing recent evidence that TSH release in vivo and in vitro is enhanced by treatment with exogenous PGs and is inhibited by drugs (e.g., indomethacin) that block PG synthesis. Pharmacological studies were then performed to test the hypothesis that hypothalamic PGs also affect TSH secretion indirectly via the appropriate hypothalamic hormones that regulate pituitary secretion. The inhibition of thyroidectomy-induced TSH secretion was used as an endpoint in choosing the best of several drugs purported to inhibit PG synthesis. The established effectiveness of indomethacin and aspirin were used for reference in testing the following drugs: naproxen, mefenamic acid, tranylcypromine, and phenelzine. Only naproxen was found to be effective, but since it was no more potent than indomethacin, the latter drug was used for subsequent work. Indomethacin was stereotaxically implanted into several hypothalamic regions known to regulate TSH secretion, and sequential plasma samples were analyzed for TSH by radioimmunoassay. Bilateral implants of indomethacin in the anterior hypothalamic area increased TSH secretion throughout the 72 hr period of study. Sham inplants at this site and indomethacin implants in other nearby sites were ineffective. These findings suggest that endogenous PGs play an inhibitory role in the hypothalamic regulation of pituitary secretion.

Inhibition of corticotropin secretion by hypothalamic administration of indomethacin.

The importance of endogenous prostaglandins (PGs) in regulating CRF release was investigated by administering the PG synthesis inhibitor, indomethacin (Ind), in specific hypothalamic regions to adult female rats. Solid Ind pellets placed in the anterior hypothalamic area (AHA) significantly reduced the elevated levels of plasma corticosterone normally observed after the surgical stress of the stereotaxic procedure. Similar pellets placed in the hypothalamic median eminence region (ME) completely blocked the normal compensatory hypertrophy observed 48 h after unilateral adrenalectomy. Plasma corticosterone secretion in response to hemorrhage (1% b.w.) or laparotomy with intestinal manipulation (LAP) in dexamethasone pretreated rats was reduced by Ind pellets, or Ind in phosphate buffer (BUF), respectively, implanted 2 h prior to the stress. These data suggest that the PGs may be important in mediating CRF release in response to a variety of stimuli.

Hypothalamic and pituitary effects of prostaglandins on ACTH secretion.

Various prostaglandins (PGs) were tested for their effects on ACTH secretion upon injection into the anterior pituitary, basomedial hypothalamus, basolateral hypothalamus, or a tail vein in anesthetized female rats. In some experiments, the PGs were injected in combination with a CRF preparation. The greatest effect seen was the stimulation of ACTH (and presumably CRF) secretion exerted at the basomedial hypothalamus. At the anterior pituitary, the PGs alone were without effect, but they did decrease the magnitude of the response to subsequent CRF.

Correlation between hypothalamic catecholamine synthesis and ether stress-induced ACTH secretion.

The rates of synthesis of norepinephrine (NE) and dopamine (DA) in several regions of the hypothalamus have been estimated after exposure of rats to stresses that increase ACTH secretion. The rate of 3H-NE and 3H-DA accumulation from 3H-tyrosine in vitro was used as an index of catecholamine synthesis rates. Exposure to ether vapor, 30 min of cold environment, or laparotomy increased ACTH secretion significantly, as indicated by plasma corticosterone levels. However, only the ether stress affected hypothalamic catecholamines; both NE and DA synthesis rates were increased in the arcuate nucleus are (ANA), but not in the median eminence (ME) or the residual hypothalamus (RH). Dexamethasone treatment blocked the stress-induced ACTH secretion, but had no affect on basal or stress-induced rates of amine synthesis. It is concluded that catecholamines in the ANA participate in mediating ether stress-induced ACTH secretion.