Iodine metabolism: preferential renal excretion of iodide derived from triiodothyronine deiodination.

Measurements were made in rats of the relative rates of accumulation in urine or in the thyroid of radioactive iodide derived from simultaneous injections of (131)I-labeled triiodothyronine and (125)I-labeled iodide. The data indicate that deiodination of triiodothyronine by the kidney results in a loss into the urine of iodine which does not enter the general body iodide pool. This renal "iodide leak" should be considered in kinetic models of iodine metabolism.

Tetraethylammonium blockade of K+ channels in GH4C1 cells regulates prolactin secretion by inducing Ca2+ influx.

Tetraethylammonium (TEA), a K+ channel blocker, induced PRL secretion and an increase in cytosol Ca2+ concentration [Ca2+]i in a dose-dependent manner between 5-20 mM in GH4C1 rat pituitary tumor-derived cells. Removal of medium Ca2+ or the addition of 1 microM nifedipine abolished both the induced [Ca2+]i increment and PRL secretion. TEA augmented the TRH-induced rise in [Ca2+]i and inhibited the rise in [Ca2+]i induced by 30 mM K+. The dynamics of TEA-, TRH- and K(+)-induced PRL secretion were different, with the TEA-induced secretory peak occurring at about 10 min compared to 2-3 min for TRH and K+. Tolbutamide, which blocks ATP-sensitive K+ channels, induced PRL secretion without causing a rise in [Ca2+]i. The results suggest that: (a) K+ channels have a complex interaction with the PRL secretory process in GH4C1 cells; (b) TEA induces PRL secretion by causing Ca2+ influx through dihydropyridine-sensitive Ca2+ channels; and (c) K+ channels play a different role in the [Ca2+]i rise induced by TRH than in that induced by depolarizing K+.

An acute release of Ca2+ from sequestered intracellular pools is not the primary transduction mechanism causing the initial burst of PRL and TSH secretion induced by TRH in normal rat pituitary cells.

With 1.5 mM [Ca2+]e, 10 nM TRH induced a prompt high-amplitude burst of hormone secretion and an initial high-amplitude [Ca2+]i burst (first phase) followed by a sustained low-amplitude [Ca2+]i increment (second phase) in both tumor-derived GH4C1 and normal adenohypophyseal (AP) cells. With less than 2 microM [Ca2+]e, in both cell types the TRH-induced first phase rise in [Ca2+]i was suppressed 30% while the second phase rise was completely abolished; however, hormone secretion was inhibited only 20-30% in GH4C1 but greater than 80% in AP cells. Thapsigargin induced a first-phase rise in [Ca2+]i in AP cells equal to that induced by 10 nM TRH but only 20% as much first-phase hormone secretion. Blocking Ca2+ channels with nifedipine inhibited TRH-induced secretion in AP cells significantly more than in GH4C1 cells. Our data indicate that the TRH-induced first-phase spike in [Ca2+]i from intracellular Ca2+ stores may play a major transduction role in hormone secretion in GH4C1 cells but not in normal AP cells. Transduction mechanisms coupled to Ca2+ influx through Ca2+ channels in the plasmalemma are apparently a much more important component of TRH-induced secretion in normal than in tumor-derived pituitary cells.

Adenosine 3',5'-cyclic monophosphate-mediated prolactin secretion in GH4C1 cells involves Ca2+ influx through L-type Ca2+ channels.

In normal anterior pituitary cells adenosine 3',5'-cyclic monophosphate (cAMP)-mediated prolactin (PRL) secretion requires Ca2+ influx. However, the role of Ca2+ in cAMP-induced secretion in the clonal rat pituitary tumor-derived GH4C1 cells remains uncertain. We examined in GH4C1 cells the effects of forskolin (FSK), an adenylate cyclase activator, and dibutyryl cAMP (DB-cAMP) on PRL secretion and intracellular Ca2+ ([Ca2+]i) dynamics. Ca2+ depletion of the medium inhibited FSK and DB-cAMP stimulated PRL secretion approximately 50%. Both FSK and DB-cAMP increased [Ca2+]i in a dose-dependent fashion. The peak amplitude in [Ca2+]i in response to each concentration of these stimuli was achieved in 3 min, corresponding to the peak PRL response to the same stimuli. Either Ca2+ depletion of the medium or addition of 2 microM nifedipine (NF) abolished the increase in [Ca2+]i caused by FSK or DB-cAMP. Our data indicate that an increase in intracellular c-AMP in GH4C1 cells produces an elevation of [Ca2+]i by opening L-type Ca2+ channels and that c-AMP-mediated PRL secretion is augmented by Ca2+ influx in GH4C1 cells as in normal pituitary cells.

Repetitive or continuous thyrotropin (TSH)-releasing hormone administration induces a biphasic rise in plasma TSH in euthyroid but not in hypothyroid rats.

In euthyroid rats, repetitive bolus injections of 0.1, 1, or 5 micrograms/100 g BW TRH every 15 min for 2 h produced a biphasic rise in the plasma TSH concentration. After an initial peak at 15 min, plasma TSH fell at 60 min to a nadir 50-80% of the 15-min peak, then rose again by 90 min to a plateau with approximately the same amplitude as the initial peak. Plasma TSH remained at this level until TRH injections were stopped at 2 h, then fell to the pre-TRH baseline by 3 h. A similar biphasic rise in plasma TSH was produced by constant infusion of 0.01, 0.1, or 1 microgram/min TRH for 3 h. If the dose of bolus TRH injected was increased at 45 min, the dip in plasma TSH at 60 min was significantly decreased. A single iv injection of 1 microgram/100 g BW T4 immediately or 4 h before the bolus TRH injections did not abolish the biphasic TSH response. However, the T4 injection 4 h before TRH significantly attenuated the amplitude of the TSH response. In hypothyroid rats, either repetitive bolus injections or constant infusion of TRH induced only a single peak of plasma TSH at 15-30 min, after which plasma TSH fell to and remained at the pre-TRH baseline. If the hypothyroid rats were injected with 2 micrograms/100 g BW T4 for 4 days before TRH bolus injections, a biphasic TSH response to continual TRH, identical to that in euthyroid rats, was produced. The pituitary TSH content of the hypothyroid rats was significantly subnormal. T4 treatment for 4 days restored both plasma and pituitary TSH levels to the euthyroid range. Our data indicate that 1) constant or repetitive exposure to TRH induces a biphasic rise in plasma TSH in euthyroid, but not in hypothyroid, rats; 2) this biphasic phenomenon is not produced by negative feedback of T4 on the thyrotroph; and 3) the rapid development of refractoriness to TRH in hypothyroid rats is not dependent on continuous exposure to a constant concentration of TRH, but may be related to the reduced TSH content of the hypothyroid thyrotroph.

Comparison of pituitary-thyroid maturation in the fetuses of rats fed an iodine-deficient or normal diet.

From the earliest detectable development of fetal pituitary-thyroid function (day 18-19 of gestation) through the first postnatal day, there was a higher degree of stimulation of the pituitary-thyroid axis in the fetuses of rats fed a low iodine diet (LID) than in those of rats fed a high iodine diet (HID). Significant differences between the two groups were consistently observed in relative thyroid size, plasma TSH, 4-h thyroid radioiodine uptake, and the labeled iodoamino acid composition of thyroid digests. Plasma T4 concentration was lower in both LID and HID fetuses and pups than in the HID mothers. Plasma T3 was not detectable (less than 20 ng/dl) in the fetuses of either group, nor was labeled T3 in the thyroid digests. Body weight, plasma T4, and pituitary TSH content were usually lower in the LID than the HID animals of comparable age; however, these differences were not consistently statistically significant (P less than 0.05). We conclude that iodine deficiency causes a marked stimulation of TSH secretion and, consequently, of thyroid growth and metabolism from the earliest development of fetal pituitary-thyroid function.

Evidence that control of fetal thyrotropin secretion is independent of both the fetal and maternal hypothalamus.

Propylthiouracil (PTU) administered to pregnant rats from day 18-21 of gestation caused a significant increase in maternal and fetal thyroid weight and plasma TSH. Fetal encephalectomy on day 18 did not significantly affect basal or PTU-stimulated pituitary-thyroid function. Destruction of the basal hypothalamus in the mother on day 13 or 16 markedly reduced maternal plasma TSH and thyroxine and prevented a PTU-induced increase in thyroid size, but did not affect fetal pituitary-thyroid function. Plasma PRL, was undetectable in both intact and encephalectomized fetuses at 21 days but was increased greater than 6-fold to approximately 2 microgram/ml in the mothers by maternal hypothalamic destruction. We conclude that fetal pituitary-thyroid function in the rat is not dependent on either fetal or maternal hypothalamic TRH.

Acute effects of hypothalamic ablation on plasma thyrotropin and prolactin concentrations in the suckling rat: evidence that early postnatal pituitary-thyroid regulation is independent of hypothalamic control.

Hypothalamic ablation was performed at various periods postnatally in animals previously administered propylthiouracil to raise plasma TSH concentrations. There was no significant change in plasma Tsh up to 8 h after hypothalamic ablation in pups 1--4 days old, whereas hypophysectomy of such pups produced a 60% fall in plasma TSH within 4 h. By the 5th postnatal day, hypothalamic ablation produced a 30% fall in plasma TSH within 4 h (P less than 0.05). By the 12th postnatal day and thereafter, the fall in plasma TSH after hypothalamic ablation was not significantly different from that seen in adults, except in 30-day-old rats in which there was a lesser effect of hypothalamic ablation on plasma TSH (P less than 0.01 in comparison to 23-day-old and adult groups). The greatest effect of hypothalamic ablation on plasma TSH was in 45-day-old animals (P less than 0.01 in comparison to adults). No significant change was produced in plasma PRL within 4 h postoperatively at any age. Our data indicate that regulation of TSH secretion in the rat is independent of hypothalamic control until after the 5th postnatal day and is fully developed by day 12. This corresponds temporally with the postnatal rise of plasma TSH, T4, and T3 and hypothalamic TRH to adult concentrations and indicates maturation of the hypothalamic regulation of TSH secretion.

Distribution of corticotropin releasing factor(s) activity in neural and extraneural tissues of the rat.

Distribution and dose-response characteristics of corticotropin releasing factor(s) (CRF) activity in the central nervous and extraneural tissues of the rat were examined with a sensitive CRF bioassay using ACTH secretion by cultured rat adenohypophyseal cells. Dose-response curves for hypothalamus were steeper than and not parallel to those for cerebral cortex, liver, serum or human urine. The minimum effective dose was smallest for the posterior pituitary. CRF activity of the basal hypothalamus was considerably higher than that in other parts of the hypothalamus, and was unaltered by 2.5 or 10 min ether stress. Our data indicate that "specific" CRF is concentrated primarily in the basal hypothalamus and posterior pituitary and is not identical with widely distributed extrahypothalamic extraneurohypophyseal CRF.

The effect of basal hypothalamic deafferentation on the nycthemeral rhythm of plasma TSH.

Basal hypothalamic deafferentation abolished the nycthemeral rhythm of both plasma TSH and corticosterone.

Studies on the heterogeneity of labeled iodoprotein from iodine-replete and iodine-deficient rats as determined by susceptibility to proteolysis.

Thyroid iodoprotein from rats fed a high-iodine diet (HID) or a low-iodine diet (LID) were labeled with radioiodine in vivo for periods ranging from 4 hr to several days. Standardized aliquots of thyroid homogenate from rats with various treatments were digested for 4 hr with 1% pancreatin or with 0.003% pancreatin after 30 min pretreatment with beta-mercaptoethanol (ME-P). Four-hr labeled iodoprotein from both LID and HID rats was equally susceptible to digestion with 1% pancreatin; however, such iodoprotein from LID rats was more susceptible to digestion with ME-P than that from HID rats. With increasing intervals up to 7 days between administering radioiodine and removing the thyroids, there was a progressive rise in the resistance to digestion in iodoprotein from LID rats, but only a slight increase in resistance in iodoprotein from HID rats. If propylthiouracil was added to the diet beginning 24 hr after radioiodine administration, there was a marked increase in the rate of development of resistance of iodoprotein to digestion. Radioautographs showed that the radioiodine was localized primarily in the peripheral follicles after 2 days PTU. Similar differences in susceptibility to digestion were found in purified thyrogobulin prepared from HID and LID rats. No change in susceptibility to digestion of thyroid iodoprotein with time after labeling was seen in hypophysectomized LID rats in which thyroid secretion and thyroglobulin turnover is known to proceed at an extremely slow rate. The data indicate that there are at least two types of iodinated thyroglobulin in the rat thyroid. One is readily susceptible to digestion and has a rapid turnover in the thyroid. The other is more resistant to digestion, has a slow rate of turnover and is located primarily in the peripheral follicles.

The importance of the hypothalamic lateral retrochiasmatic area in the control of adrenocorticotropin and thyrotropin secretion.

Anterolateral hypothalamic deafferentiation was made in rats to explore the importance of the neural pathways through the lateral retrochiasmatic area (RCAL) in the regulation of ACTH and TSH secretion. In rats with complete bilateral RCAL transection, pituitary-adrenal function was altered in the following respects compared to sham-operated controls. 1) Basal plasma ACTH, corticosterone (B), and adrenal weight were depressed. 2) Plasma ACTH and B elevation in response to 3-min ether inhalation were markedly decreased or abolished. 3) Insulin-induced hypoglycemia produced no or little plasma B elevation. 4) Lysine-vasopressin was significantly less effective in inducing pituitary-adrenal activation. Reductions in plasma ACTH and B concentrations and adrenal weight were correlated with the completeness of the RCAL transection. The plasma TSH concentration was lower in the deafferented rats than in the controls regardless of the completeness of the cut at the RCAL, indicating that the neural pathways traversing this area do not possess a critical importance for the regulation of TSH if the rest of the hypothalamus is deafferented anterolaterally. We conclude that intact neural connections between the medial basal hypothalamus and the central nervous system at the RCAL are essential for the maintenance of normal hypothalamic-pituitary-adrenal function.

Thyrotropin secretory response to thyrotropin-releasing hormone in the hypothyroid perinatal rat: further evidence of thyrotroph independence of the hypothalamus during early ontogenesis.

Propylthiouracil fed to pregnant rats for the last week of gestation to induce maternal and fetal hypothyroidism induced a 3-fold rise in plasma TSH concentration in the newborn pups compared to a 4-fold rise in their mothers. Subcutaneous administration of 1 ng/g BW TRH caused a greater rise in plasma TSH in the hypothyroid pups than in their mothers. These results, in combination with published data, indicate that the apparent independence of pituitary-thyroid function from TRH control during early ontogenesis in the rat is primarily due to delayed maturation of the hypothalamic TRH system.

Delineation of the hypothalamic area controlling thyrotropin secretion in the rat.

Discrete midline hypothalamic lesions were made in male rats in the region of the paraventricular nuclei (PVN), ventromedial nuclei, and medial preoptic area (mPO) using modified Halasz C-shaped knives. In euthyroid rats, small lesions, including the PVN and little surrounding tissue, or large lesions, including portions of the dorsomedial nucleus, anterior hypothalamus, and preoptic area in addition to the PVN, caused a similar 60% drop in the plasma TSH concentration within 2 days which persisted for at least 3 weeks. PVN lesions also produced a significant decrease in plasma TSH in hypothyroid rats and diminished both the increase in plasma TSH in response to thyroidectomy and the decrease induced by ether inhalation. Ventromedial nuclei lesions preserving the PVN inconsistently decreased plasma TSH. mPO lesions anterior to the PVN induced a transient elevation of plasma TSH and GH only in hypothyroid rats. TRH-stimulated TSH secretion was not affected by any of the lesions. The results suggest: 1) the PVN and their immediate vicinity are of primary importance for maintaining a normal TSH response to the stimuli investigated, and 2) the mPO area tonically inhibits TSH secretion, presumably through its role in somatostatin secretion.

The effect of basal hypothalamic isolation on pituitary-thyroid activity and the response to propylthiouracil.

Basal hypothalamic deafferentation extending from the posterior border of the optic chiasm to the mid-mammillary bodies resulted in depression of plasma TSH, thyroxine (T4), and triiodothyronine (T3) concentration to 50% of normal controls within 7 days. Administration of 0.15% propylthiouracil (PTU) in the diet form postoperative day 26 caused a pronounced drop in the plasma T3 level and a rise in plasma TSH level within two days in the control animals, but had little effect during this interval in the deafferented animals. After 12 days of PTU, plasma T3 and T4 concentrations had dropped to undetectable concentrations in the control animals but both were still detectable in the deafferented animals. After 25 days of PTU, plasms T4 and T3 levels were undetectable and plasma TSH levels were significantly elevated above normal in all animals. Thyroid hypertrophy at that time was as great in the deafferented as in the control rats, although plasma TSH concentration was 50% lower in the former. Administration of 0.1 mug/100 g BW TRH iv on postoperative day 37, when plasma T4 and T3 were undetectable in the controls but still present in the deafferented animals, produced an equally high concentration of plasma TSH in all animals. We interpret these data to support the concepts that: 1) a major source of neural drive of that TRH which stimulates the secretion of TSH by the adenohypophysis lies outside the medial basal hypothalamus, 2) a decrease in TRH reaching the adenohypophysis causes a lower setting of the "thyrostat" sensitive to the concentration of circulating thyroid hormone, and 3) increased TSH secretion and resultant goitrogenesis is delayed in animals with impaired TRH secretion because of the slower rate of secretion of thyroid hormone than in intact controls and the longer time thus required to markedly reduce the concentration of circulating thyroid hormone.

Studies on the corticotrophin-releasing activity of vasopressin, using ACTH secretion by cultured rat adenohypophyseal cells.

CRF activity of synthetic vasopressins and pitressin was studied in an in vitro system of cultured rat adenohypophyseal cells using direct measurement of ACTH by radioimmunoassay. Pitressin (posterior pituitary extract) induced a dose-related secretion of ACTH whereas synthetic arginine or lysine vasopressin were devoid of CRF activity, even with the largest tested dose (4 mug/ml). No potentiation of the CRF activity of hypothalamic extract was observed with any vasopressin preparation studied. We concluded that: 1) the CRF activity of posterior pituitary extract is not due to vasopressin, and 2) the ACTH secretion induced by vasopressin administration in vivo is unlikely to be due to a direct effect of vasopressin on adenohypophyseal cells.

Evidence that the hypothalamus mediates endotoxin stimulation of adrenocorticotropic hormone secretion.

The site of action of Escherichia coli endotoxin in inducing ACTH secretion was studied in vivo and in vitro. Hypophysectomized rats, bearing two to three transplanted pituitaries under the kidney capsule and "primed" with exogenous ACTH, responded to 2.0-7.5 microgram/100 g BW ip or iv endotoxin with a several-fold increase of plasma corticosterone. This response was markedly reduced by hypothalamic lesions and completely abolished by removing the entire forebrain. Endotoxin added directly to cultured rat adenohypophyseal cells in a concentration up to 10 microgram/ml did not induce significant ACTH secretion. We conclude that endotoxin-induced ACTH secretion from heterotopically transplanted pituitaries is mediated primarily by the hypothalamus, presumably through hypothalamic CRF that reaches the transplanted pituitaries via the systemic circulation.

There is a nyctohemeral rhythm of type II iodothyronine 5'-deiodinase activity in rat anterior pituitary.

We found a nyctohemeral rhythm of type II iodothyronine 5'-deiodinase (5'-D-II) with a zenith at midnight in rat anterior pituitary, but not in brown adipose tissue. There was no nyctohemeral rhythm of 5'-D-I in anterior pituitary, liver, or kidney. Hypothyroidism abolished the nyctohemeral rhythmicity in anterior pituitary 5'-D-II. The rhythmicity of anterior pituitary 5'-D-II may play a role in setting the nyctohemeral rhythm of TSH secretion by regulating the degree of negative feedback by locally generated T3 in the thyrotroph.

Rhythmicity of triiodothyronine generation by type II thyroxine 5'-deiodinase in rat pineal is mediated by a beta-adrenergic mechanism.

Type II T4 5'-deiodinase plays an essential role in converting T4 to T3 in extrathyroidal tissues, thus allowing the full development of a cellular effect of thyroid hormone. This enzyme is one of the most important factors in regulating local action of thyroid hormone. Its activity in the rat pineal was 20-30 times higher at midnight than at noon; this nocturnal rise was abolished by the beta-adrenergic blocker, propranolol. The beta-agonist isoproterenol, caused a 3-fold increase in diurnal enzyme activity. There is thus a marked nyctohemeral variation in T4 activation in the pineal, probably mediated by a beta-adrenergic mechanism. Since the temporal peak of pineal T4 activation corresponds to that of pineal function, our data suggest a previously unrecognized role of thyroid hormone in pineal regulation.

A sensitive and simple in vitro assay for corticotropin-releasing substances utilizing ACTH release from cultured anterior pituitary cells.

Graded doses of rat hypothalamic extract (HE) were added to dishes containing dispersed, pooled rat adenohypophyseal cells cultured for several days. ACTH secretion into the medium gave a linear log-dose response curve over a 100-fold range between 0.01 and 1 mg of NIH-HE (0.0125-1.25 rat hypothalamus). Forty percent of the maximal ACTH secretion in response to a given dose of HE occurred within 3 min. No decrease in intracellular ACTH occurred at any time or with any dose of HE, indicating that secretion was always balanced by production. ACTH secretion stopped as soon as HE-containing medium was replaced by medium without HE. The same cultured cells could be satisfactorily used in repetitive assays performed on the same or different days.