George A. Hedge

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.

The roles of the opioid peptides in controlling thyroid stimulating hormone release

We have studied the role of the opioid peptides in controlling TSH secretion. Morphine sulfate significantly decreased, while naloxone had no effect on, basal plasma TSH levels of female rats. In contrast, naloxone blocked the stress-induced fall in plasma TSH. Microinjection of beta-endorphin into the third ventricle resulted in a fall in TSH while such injection of naloxone into the posterior hypothalamus increased TSH. Microinjection of beta-endorphin directly into the pituitary caused a rise in plasma TSH. It is concluded that opioid peptides probably play no role in basal TSH secretion, but are involved in the stress-induced fall in TSH. Furthermore, it appears that opioid peptides have a site of action in the hypothalamus to decrease TSH and a direct pituitary action to increase TSH.

Dynamics and Regulation of TSH Secretion by Superfused Anterior Pituitary Cells

Superfused dispersed cells respond rapidly to 2- to 10-min pulses of TRH (10(-10) to 10(-7) M) in a dose-dependent manner. The effects of decreasing the stimulus duration can be overcome by a proportional increase in concentration of TRH. A TRH stimulus of 10 min or greater duration results in a sharp peak in TSH secretion followed by a lower plateau. Somatostatin (10(-8) M inhibits the response to TRH (t X 10(-9) M). T3 (2.0 microgram/dl) inhibits TRH-induced TSH secretion by superfused pituitary fragments, but not by dispersed cells. Corticosterone (50 microgram/dl), however, inhibits crude CRF-induced ACTH secretion by such cells.

Thyroid hormones are required for daily rhythms of plasma corticosterone and prolactin concentration

Abstract Daily patterns of plasma corticosterone (B) and prolactin (PRL) concentration were measured in female rats which were intact (INTACT), thyroidectomized (THYREX), or thyroidectomized and given thyroxine (T 4 ) replacement (THYREX + T 4 ) (20 μg T 4 /day). Bimodal daily rhythms of plasma B were present in INTACT rats and THYREX + T 4 rats. However, no plasma B rhythm was detectable in THYREX rats. THYREX + T 4 rats, which had greater mean T 4 concentrations than INTACT rats (6.0 μg/dl vs. 3.5 μg/dl), had a plasma B rhythm of greater amplitude than INTACT rats. Plasma PRL rhythms were detectable in INTACT and THYREX + T 4 rats, but not in THYREX rats. INTACT rats had a single peak in plasma PRL, whereas three plasma PRL peaks were present in THYREX + T 4 rats. It was concluded that thyroid hormones are required for the expression of plasma B and PRL rhythms and that the levels of thyroid hormones can alter the amplitude of the B rhythm and the shape of the PRL rhythm.

Sympathetic thyroidal vasoconstriction is not blocked by a neuropeptide Y antagonist or antiserum

Sympathetic nerve fibers to thyroid blood vessels contain both norepinephrine (NE) and neuropeptide Y (NPY). To assess the involvement of endogenous NPY in the sympathetic neural control of thyroid blood flow, appropriate doses of a selective NPY antagonist, alpha-trinositol, and an NPY antiserum (NPY-AS) were used during cervical sympathetic trunk stimulation in anesthetized rats. During all experiments, thyroid blood flow was continuously monitored by laser Doppler blood flowmetry. Neither alpha-trinositol nor NPY-AS blocked the thyroidal vasoconstriction evoked by either the first or second stimulation of the cervical sympathetic trunks. Our results suggest that NPY is not involved either directly or indirectly during acute sympathetic vasoconstriction in the rat thyroid gland.

Factors Involved in the Attenuation of the TSH Response to a Second Injection of TRH in the Rat

The attenuation of thyrotropin (TSH) responsiveness to a second injection of thyrotropin-releasing hormone (TRH) was studied in adult female rats. This attenuation occurs using submaximal doses of TRH and it is proportional to the dose of the first stimulus within the range of 0-1,250 ng TRH/100 g BW. The magnitude of the attenuation varies inversely with the interstimulus interval whether the TRH is administered intravenously or directly into the anterior pituitary. This diminished responsiveness is independent of stress or anesthesia, and it is not due to negative feedback by thyroid hormones released in response to the initial TRH stimulation. Exogenous rat TSH given to mimic the response to TRH also results in attenuation, and it is suggested that TSH is at least in part responsible for the attenuation observed after TRH administration.

Helodermin, but not cholecystokinin, somatostatin, or thyrotropin releasing hormone, acutely increases thyroid blood flow in the rat.

In the present study, we investigated whether peptides located within the thyroid gland, but not directly found in nerve fibers associated with blood vessels, might influence thyroid blood flow. Specifically, we evaluated the effects of helodermin, cholecystokinin (CCK), somatostatin (SRIF) and thyrotropin releasing hormone (TRH) given systemically on thyroid blood flow and circulating thyroid hormone levels. Blood flows in the thyroid and six other organs were measured in male rats using 141Ce-labeled microspheres. Circulating thyrotropin (TSH) and thyroid hormone levels were monitored by RIA. Helodermin (10(-10) mol/100 g BW, i.v. over 4 min) markedly elevated thyroid blood flow (52 +/- 6 vs. 10 +/- 2 ml/min.g in vehicle-infused rats; n = 5). Blood flows to the salivary gland, pancreas, lacrimal gland and stomach (but not adrenal and kidney) were also increased during helodermin infusions. CCK, SRIF, and TRH were without effect on blood flows to the thyroid and other organs even though these peptides were tested at higher molar doses than helodermin. Helodermin, CCK, or SRIF did not affect thyroid hormone or plasma calcium levels. As expected however, plasma TSH and T3 levels were increased at 20 min and 2 h, respectively, following TRH infusions. Since helodermin shares sequence homology with VIP, we next compared the relative effects of these two peptides on thyroid and other organ blood flows. VIP (10(-11) mol/100 g BW, i.v.) was more potent in increasing blood flows to the thyroid, salivary gland, and pancreas than an equimolar dose of helodermin. This study shows that while helodermin, like VIP, has the ability to increase thyroid and other organ blood flows, it appears to be a less potent vasodilator.

Neuropeptide control of thyroid blood flow and hormone secretion in the rat

The effects of peptide HI (PHI), neuropeptide Y (NPY), and substance P (SP) on thyroid blood flow and hormone levels were studied in anesthetized rats. Regional blood flows were determined using radioactive microspheres. No change in heart rate or mean left ventricular pressure occurred during these neuropeptide infusions (0.625 micrograms iv over 2 min). PHI treatment resulted in a four-fold increase in thyroid blood flow. Blood flows to the pancreas and salivary gland also increased during PHI treatment. Infusions of NPY or SP did not significantly alter thyroid blood flow. However, SP decreased blood flow to the spleen and small intestine. These neuropeptides had no effect on blood flows to the adrenal, kidney, brain, heart, and adipose tissues. Following PHI, NPY, and SP infusions, plasma triiodothyronine and thyroxine levels were not different from values in saline-treated rats. This study demonstrates that PHI, like vasoactive intestinal peptide, is a potent thyroidal vasodilator at a dose that does not affect circulating thyroid hormone secretion.

Vasoactive Intestinal Peptide Enhances Thyroidal Iodide Uptake During Dietary Iodine Deficiency

The presence of vasoactive intestinal peptide and neuropeptide Y in thyroid nerves and their effects on thyroid blood flow are well known. However, the effects of these two neuropeptides on the various processes involved in thyroid hormone biosynthesis and release have not been fully explored. We have now tested these two peptides for effects on an early step in thyroid hormone biosynthesis, namely iodide uptake, a process which is comprised of trapping and organification. In these experiments, we have used anesthetized adult male rats pretreated with thyroxine or fed a low iodine diet to increase thyroidal sensitivity. Vasoactive intestinal peptide significantly increased iodide uptake in rats fed an iodine deficient diet but not in those fed a normal iodine diet. This effect disappeared if animals were pretreated with propylthiouracil. Neuropeptide Y did not alter iodide uptake in rats on either the low or the high iodine diet, regardless of the presence or absence of propylthiouracil. The effect of vasoactive intestinal peptide on iodide uptake could be due to its influence on the organification of iodine, or on thyroid blood flow, or on both processes.

Expression of preproNPY and precursor VIP mRNAs in rats under hypo- or hyperthyroid conditions.

Both neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) are present in thyroid nerves and have been shown to alter thyroid activity. The present study was conducted to determine whether hypo- or hyperthyroidism is associated with changes in the expression of the mRNAs for these neuropeptides in the major ganglia which supply nerves to the thyroid or within the thyroid gland itself. Hypo- or hyperthyroid conditions were induced by the administration of propylthiouracil (PTU) or thyroxine (T(4)), respectively, for 6 days. Control rats received vehicle injections. Total RNA from superior cervical ganglia (SCG), local thyroid ganglia, thyroid gland, and selected other tissues was extracted and mRNA levels were analyzed using Northern blot procedures. No significant changes in preproNPY or precursor VIP mRNA levels were detected in the SCG or the local thyroid ganglia in response to PTU or T(4) treatment. However, PTU treatment was associated with an increase in preproNPY mRNA levels in the thyroid gland itself. These results indicate that changes within the thyroid axis in response to these hypo- and hyperthyroid conditions do not include alterations in steady-state preproNPY or precursor VIP mRNA concentrations in the major ganglia which supply nerves to the thyroid gland. However, intrathyroidal preproNPY mRNA levels are increased as a consequence of the thyroidal adaptation to a PTU challenge.