Locus coeruleus lesions decrease norepinephrine input into the medial preoptic area and medial basal hypothalamus and block the LH, FSH and prolactin preovulatory surge.

The aim of this work was to study the role of the dorsal noradrenergic ascending pathway (DNAP), which originates in the locus coeruleus (LC) on the preovulatory surge of luteinizing hormone (LH) follicle-stimulating hormone (FSH) and prolactin (PRL) by producing bilateral electrolytic lesions (cathodal or anodal) in this nucleus. LC lesions were placed at 11.00 h on proestrus in female rats with regular 4-day estrous cycles. Intact rats, sham-operated as well as animals with missed lesions served as controls. In Experiment I, anodal current was applied and hourly blood samples were withdrawn (from 13.00 to 17.00 h) via a jugular catheter from conscious, freely moving rats for determination of plasma LH, FSH and PRL concentrations. In Expt. II, Expt. I was repeated using cathodal current and collecting blood samples hourly from 13.00 to 18.00 h. In both experiments the animals were sacrificed on the next morning when the occurrence of ovulation was checked. The medial septal area (MSA), medial preoptic area (MPOA), and medial basal hypothalamus (MBH) were dissected and assayed for norepinephrine (NE), dopamine (DA) and 5-hydroxyindoleacetic acid (5-HIAA) content. Experiment III was performed in order to test if a hormonal discharge occurred immediately after lesion placement. Blood samples were collected immediately before and 15, 30, 60 and 90 min postoperatively (from 11.00 to 12.30 h). Either anodal or cathodal lesions blocked the proestrous surge of LH, FSH and PRL. The hypothesis that the lesions advanced or delayed these hormonal surges was rejected since we found no increases in the hormonal levels from 11.00 to 12.30 or from 13.00 to 18.00 h, and ovulation was not observed on the following morning in the lesioned animals. Since control, sham-operated and missed-lesion groups exhibited LH, FSH and PRL surges and ovulation, this blockage appears to be caused by the destruction of the LC neurons. Also, this blockade was correlated with a decrease in the NA content in the MPOA and MBH, but not in the MSA, whereas the DA and 5-HIAA content were not changed in all groups examined. The results lead us to suggest that the integrity of noradrenergic afferent input from the LC to luteinizing hormone-releasing hormone neurons in the MPOA and MBH is essential for triggering the preovulatory surge mechanisms for gonadotrophins and PRL.

Interaction between norepinephrine and serotonin in the neuroendocrine control of growth hormone release in the rat.

Both alpha 2-adrenergic (alpha 2) and serotonergic (5HT) neurons are associated with stimulation of GH secretion via GRH release. The object of this study was to determine whether the 5HT system is involved in the stimulation of GH secretion by alpha 2-receptor agonists. There are two parts of this study. In the first, the relationship between alpha 2-5HT systems were analyzed by determining if alpha 2-stimulated GH release is mediated by 5HT. In this model, systemically administered alpha 2-agonists [clonidine (CLON) or UK14,304] were tested against 5HT antagonists (meterogoline or cyproheptadine) or 5HT synthesis inhibitors (p-chlorophenylalanine methylester hydrochloride). In the second, sites of 5HT-GRH interaction were determined by testing the response to CLON after 5HT neurotoxin [5,7-dihydroxytryptamine (5,7-DHT)] microinjection at specific hypothalamic nuclei. In both experiments sequential blood samples were withdrawn from silastic jugular cannulas in unanesthetized, freely moving animals. Metergoline (0.045 and 0.135 mg/kg, iv) and cyproheptadine (0.969 micrograms/kg, iv) suppressed, in a dose-dependent manner, the CLON (33 or 66 micrograms/kg, iv)-induced GH surge that was detected 15-30 min after injection in control animals. Both cyproheptadine (0.969 micrograms/kg, iv) and p-chlorophenylalanine (300 mg/kg, ip) effectively suppressed the UK14,304 (220 micrograms/kg, iv)-induced GH surge that occurred 15-30 min after injection in control animals. These data suggest that an intact 5HT system is required for alpha 2-stimulated GH release. 5,7-DHT neurotoxin microinjected into the midline arcuate nucleus (6 micrograms/mg,iv) or bilaterally into the ventromedial nucleus or perifornical area (4 micrograms/0.2 microliter) 5 days previously suppressed the CLON (30 micrograms/kg)-induced GH surge only in animals with arcurate nucleus lesions. To determine if the suppression was mediated by inhibition of GH-releasing hormone (GRH) or stimulation of somatostatin (SRIF), an additional experiment was conducted including 5,7-DHT arcuate nucleus-lesioned animals injected with anti-SRIF. Inasmuch as anti-SRIF failed to reverse the 5,7-DHT suppression of GH secretion, the results of this experiment suggest that GRH mediates NE-5HT-induced GH secretion. In conclusion, these data suggest that alpha 2 activation of GH secretion requires intact serotonergic terminals in the arcuate nucleus and most likely involves GRH rather than SRIF, release.

The paradox of alpha 2 adrenergic regulation of prolactin (PRL) secretion. I. The PRL-releasing action of the alpha 2 receptor agonists.

Systemic (IV) administration of the alpha 2 receptor agonist clonidine is known to stimulate secretion of PRL and growth hormone (GH) suggesting a stimulatory role of the central alpha 2 receptors in the regulation of the two hormones. The present work confirms this notion for GH but indicates that the alpha 2 agonists stimulate PRL release by a peripheral action not involving central alpha 2 receptors. This conclusion is based on the following findings: 1) The minimum effective IV dose of clonidine or UK 14304 was four times larger for activation of PRL than GH secretion and had already manifest extracentral effects (elevation of arterial BP). 2) Subcutaneous injection of UK 14304 (220 micrograms/kg) elevated plasma GH but not PRL levels indicating that an effective activation of the central alpha 2 receptors does not stimulate PRL release. 3) Peripherally acting alpha 2 agonists (p-aminoclonidine, oxymetazoline) had no effect on GH secretion but stimulated PRL release in a manner identical with the effect of clonidine or UK 14304. 4) The peripherally acting alpha 2 antagonist DG-5128 blocked only the PRL secretory response to UK 14304 whereas the peripherally and centrally active yohimbine blocked the PRL and GH responses.

The paradox of alpha 2 adrenergic regulation of prolactin (PRL) secretion. II. PRL-releasing action of the alpha 2 receptor antagonists.

It has been suggested that the stimulation of the secretion of PRL by the alpha 2 adrenergic receptor antagonists (yohimbine, piperoxane) results from blockade of an inhibitory influence imposed on PRL release by the central alpha 2 receptors (7, 15). Our present results do not support these conclusions for the following reasons: 1) The effectiveness of the alpha 2 receptor antagonists yohimbine (YOH), rauwolscine (RAU), Wy 26392 and idazoxan (IDAZ) respectively to activate secretion of PRL was not related to their alpha 2 antagonist potencies. RAU was more effective in activation of PRL secretion than either YOH or Wy 26392 although it had a similar alpha 2 antagonist activity, while IDAZ, the most potent alpha 2 blocker among the four compounds, did not stimulate PRL secretion. 2) The PRL-releasing effect of YOH or Wy 26392 was reversed by the alpha 2 agonist clonidine but the same effect of RAU was not, speaking against a common alpha 2-mediated mechanism of action of the three antagonists. 3) The PRL-stimulating effect of YOH, RAU or Wy 26392 persisted following inhibition of NE synthesis and presumably release with FLA 63, DDC or combination of reserpine and DDC. 4) Conversely, we found no indication for an inhibiting influence of activation of the alpha 2 receptors on the secretion of PRL. We conclude that the stimulation of PRL secretion by the alpha 2 receptor antagonists is not derived from blockade of the central alpha 2 receptors but from other, not yet defined properties of the drugs.

Evaluation and characterization of the hyt/hyt hypothyroid mouse. II. Abnormalities of TSH and the thyroid gland.

The hyt/hyt mouse (BALB/cBY-hyt, C.hytRF) provides a useful model for exploring the effect of inherited severe primary hypothyroidism. Studies were undertaken to try to define the basis of the primary hypothyroidism in mice homozygous for the autosomal recessive gene, hyt. These mice had congenital hypothyroidism of fetal onset after 15 days post conception. Through their lifetime, the hyt/hyt mice had reduced serum thyroxine (T4), triiodothyronine (T3), reduced thyroid gland intralumenal colloid on electron microscopy and a 100-fold elevation of TSH-like activity compared to hyt/+ littermates. Thyroglobulin made in hyt/hyt animals was similar in size to normal thyroglobulin which was inconsistent with a major structural thyroglobulin gene defect. The thyroglobulin was iodinated. Marked, erratic dilation of rough endoplasmic reticulum (RER) was noted in hyt/hyt mouse follicular cells. Despite these ultrastructural findings, pulse chase and immunoprecipitation studies with isolated hyt/hyt and normal thyroid glands indicated that normal thyroglobulin processing occurred in the RER and Golgi of the hyt/hyt mice. The hyt/hyt thyroid glands were hypoplastic compared to hyt/+ littermates. Histologically, the hyt/hyt thyroid glands demonstrated an increase in smaller follicular cells, and greater variability in follicular size compared to hyt/+ littermates. Histological and ultrastructural abnormalities in the gland were similar to those seen in certain cases of human congenital hypothyroidism with TSH receptor insensitivity of the thyroid gland. These findings along with the significant TSH elevation, the reduction in colloid and in serum T3 and T4, the efficacy of the hypothalamo-pituitary-thyroid feedback system, and previous observations of reduced iodine uptake and intrathyroidal T4, suggested that primary hypothyroidism in the hyt/hyt mouse might be due to a defect in TSH responsivity of the thyroid gland.

Prolactin (PRL) release-inhibiting properties of the alpha 2 adrenergic receptor antagonist idazoxan: comparison with yohimbine.

The effects of the alpha 2 adrenergic receptor antagonists yohimbine (YOH) and Idazoxan (ID) on secretion of PRL were compared in nonanesthetized male rats bearing permanent intraatrial cannulae for i.v. drug delivery and serial blood sampling. YOH induced a dose-related elevation of basal plasma PRL levels. ID had either no effect or a tendency to lower them and effectively inhibited stimulation of PRL secretion with morphine, 5-hydroxytryptophan (5HTP), quipazine or restraint stress. YOH at low doses did not alter the PRL secretory responses to these stimuli or enhanced them at the highest dose used (1.56 mg/kg). ID inhibited the PRL-stimulating, effect of 5HTP or morphine following inhibition of NE synthesis with FLA63 or pretreatment with clonidine. It also blocked the effect of quipazine in rats pretreated with prazosin. It is concluded that ID, in a complete contrast to YOH effectively inhibits PRL secretion. The inhibitory mechanism appears to be unrelated to its interaction with the alpha adrenergic receptors.

Opioid kappa receptors and the secretion of prolactin (PRL) and growth hormone (GH) in the rat. I. Effects of opioid kappa receptor agonists bremazocine and U-50,488 on secretion of PRL and GH: comparison with morphine.

The effects of bremazocine and U-50,488, two selective opioid kappa receptor agonists, and the preferential mu receptor agonist morphine on the secretion of PRL and GH were compared in conscious male rats bearing permanent right atrial cannulae for serial blood sampling and drug delivery. All three opioids stimulated PRL secretion in a dose-related manner, but the kappa agonists differed from morphine in several respects. They were considerably more potent than morphine in triggering a PRL response, but were unable to elevate PRL levels to more than 100 ng/ml, whereas morphine, at the highest dose (4.5 mg/kg), induced an almost twice larger response. Also their PRL-releasing effect was inhibited more strongly by the preferential kappa receptor antagonist Mr-2266 than by naloxone, whereas Mr-2266 and naloxone, which are equipotent as antagonists of the mu receptors, were equipotent in suppressing the PRL-stimulating effect of morphine, a mu agonist. In a complete contrast to morphine, which effectively stimulated GH secretion, the kappa agonists had no effect on GH release at lower doses and suppressed it at higher doses. It is concluded that the PRL-releasing effect of the kappa agonists is mediated by the kappa receptors which may participate with the mu receptors in regulation of PRL secretion by opioids. The GH-inhibiting effect of the kappa agonists requires further clarification.

Opioid kappa receptors and the secretion of prolactin (PRL) and growth hormone (GH) in the rat. II. GH and PRL release-inhibiting effects of the opioid kappa receptor agonists bremazocine and U-50,488.

An analysis of the GH release-inhibiting action of the opioid kappa receptor agonists bremazocine and U-50,488, established earlier, was attempted by testing the agonists against activation of GH secretion by morphine or clonidine in male rats bearing right atrial cannulae for serial blood sampling and drug delivery. Both kappa agonists inhibited the effect of subsequent administration of clonidine in a dose-related manner. Bremazocine was approximately ten times more potent than U-50,488, a ratio corresponding to the known affinities of the two compounds for the kappa receptors. The inhibiting action of bremazocine was more strongly reversed by the preferential kappa receptor antagonist Mr-2266 than by naloxone, neither of which interfered with the GH-stimulating effect of clonidine. Bremazocine, however, did not alter the activation of GH secretion by exogenous growth hormone releasing factor. Thus, the inhibiting effect of bremazocine and probably U-50,488 seems to be derived from stimulation of the kappa receptors which in turn activates a GH release inhibiting mechanism of unknown identify which, however, does not involve release of somatostatin. Both kappa agonists also inhibited the effect of morphine, but in this case U-50,488 was approximately hundred times less effective than bremazocine. Since bremazocine and U-50,488 are antagonists of the delta receptors, which seem to mediate the GH-releasing effect to morphine, their inhibiting effect in this instance may be related to this property rather than to an action on the kappa receptors. Bremazocine, but not U-50,488, was also highly effective in inhibiting stimulation of PRL secretion by morphine.(ABSTRACT TRUNCATED AT 250 WORDS)

On the mechanism of growth hormone autofeedback regulation: possible role of somatostatin and growth hormone-releasing factor.

To determine the role of somatostatin (SRIF) and GH-releasing factor (GRF) in GH autofeedback, 20 micrograms rat GH in 2 microliter were injected into the third ventricle (IVT) 1 or 3 h before injection of the alpha 2-receptor stimulator clonidine (50 micrograms/kg, iv), which elevates plasma GH and TSH levels in normal rats. GH preinjected 1 or 3 h before clonidine significantly suppressed the clonidine-induced GH surge, whereas TSH release was not affected by GH. Preinjection of ovine LH IVT following the same protocol did not inhibit the clonidine-induced GH surge, suggesting a specific effect of IVT GH. Passive immunization with 400 microliters sheep antisomatostatin serum did not reverse the inhibition of the clonidine-induced GH surge by exogenous GH administered IVT either 1 or 3 h before clonidine. The TSH response was augmented by this procedure. Furthermore, IVT GH did not reduce the surges of GH and TSH elicited by GRF (250 ng/kg, iv) and TRH (150 ng/kg, iv) administered 1 or 3 h after IVT rat GH. These results suggest that GH autofeedback is mediated by reduced GRF secretion, rather than enhanced SRIF release.

Possible delta receptor mediation of the effect of beta-endorphin on luteinizing hormone (LH) release, but not on prolactin (PRL) release, in the ovariectomized rat.

Administration of opioid receptor antagonists was utilized to determine the opioid receptor type involved in the suppression of LH release by beta-endorphin (beta-END). Long-term (three to four weeks) ovariectomized rats with chronic third ventricular cannulae were fitted with jugular catheters and received treatment with vehicle or one of three opioid antagonists. The delta antagonist ICI 154, 129, but not the mu1 or mu antagonists naloxazone or beta-funaltrexamine, respectively, blocked the suppressive effect of beta-END on plasma LH levels and transiently but significantly increased LH levels above preinfusion value. None of the antagonists significantly reduced the beta-END-induced release of PRL. These results provide evidence that the inhibitory effect of beta-END on LH release may be mediated by delta receptors.

Differential role of the opioid mu and delta receptors in the activation of prolactin (PRL) and growth hormone (GH) secretion by morphine in the male rat.

Administration of naloxazone (50 mg/kg i.v.), an irreversible, selective and long acting antagonist of the mu 1 subclass of the opioid receptors, strongly reduced stimulation of PRL secretion by morphine (5.0 mg/kg i.v.) injected 24 hours later into conscious, unrestrained rats. In contrast, the effect of morphine on PRL release was unimpaired in rats treated 24 hours beforehand with either the reversible opioid antagonist naloxone (50 mg/kg i.v.), or the vehicle for naloxazone. A complete suppression of the PRL response to morphine (3.0 mg/kg i.v.) was observed in animals given intraventricular (IVT) injection of beta- funaltrexamine (beta-FNA, 2.5 micrograms), another selective, irreversible and long acting antagonist of the mu receptors, 24 hours beforehand. Neither naloxazone nor beta-FNA had any effect on the activation of GH secretion by morphine, which, however, was conspicuously reduced by ICI 154, 129, a preferential delta receptor antagonist, injected IVT (50 micrograms) 5 minutes before morphine. ICI 154, 129 had no effect on the PRL response to morphine. It is concluded that the PRL stimulating effect of morphine is mediated by the mu receptors, whereas activation of GH probably involves the delta sites.

Site of action for beta-endorphin-induced changes in plasma luteinizing hormone and prolactin in the ovariectomized rat.

A number of sites have been hypothesized as loci at which opioid substances act to alter the secretion of luteinizing hormone (LH) and prolactin (PRL) (1-8). The aim of the present study was to determine the site(s) at which the opioid peptide beta-endorphin (beta-END) acts to influence plasma LH and PRL levels in the ovariectomized (OVX) rat. beta-END, administered into the third ventricle of conscious OVX rats fitted with jugular catheters, significantly decreased plasma LH in doses greater than or equal to 50 ng and increased PRL levels at all doses administered (10, 50, 100 and 250 ng) in a dose dependent fashion. To identify possible central nervous system sites of action, 250 ng beta-END was unilaterally infused into various brain sites. Plasma LH was significantly decreased and plasma PRL significantly increased by infusions into the ventromedial hypothalamic area, the anterior hypothalamic area, and the preoptic-septal area. There was no significant effect of beta-END infusions into the lateral hypothalamic area, amygdala, midbrain central gray, or caudate nucleus. When hemipituitaries of OVX rats were incubated in vitro with beta-END (10(-7)M to 10(-5)M), there was no suppression of basal or LHRH-induced LH release, nor was there any alteration of basal PRL release. It is concluded that beta-END acts at a medial hypothalamic and/or preoptic-septal site and not the pituitary, to alter secretion of LH and PRL.

Site of thyroxine-evoked decrease of jejunal lactase in the rat.

Localization of thyroid-mediated decrease of lactase activity along the villus-crypt unit in adult rat jejunum was studied 24 and 48 h after first injection of L-thyroxine (200 micrograms/100 g body wt) every 24 h. [3H]thymidine was also given at time of first thyroxine injection. Serum thyroid-stimulating hormone, food intake, and body weight were significantly decreased within 24 h. Total jejunal protein and villus-crypt height were unchanged during the time period studied. Lactase activity (expressed both as per tissue protein and per intestinal segment) was significantly decreased in jejunum and midjejunum within 24 h. Serial sectioning of the jejunal villus-crypt unit in a cryostat showed that the site of decrease in lactase activity at 24 h was in the apical villus and by 48 h extended along the entire height of the villus. Epithelial cell migration measured both by histoautoradiography and scintillation counting of [3H]thymidine in cryostat sections revealed no difference between control and thyroid-treated animals at both 24 and 48 h. The decrease in lactase activity at 24 h was in advance of the leading edge of radioactivity, indicating that the thyroid-evoked response occurred in mature enterocytes already on the villus.

On the prolactin-inhibiting effect of neurotensin. The role of dopamine.

Neurotensin (NT) when injected in a dose of 5 micrograms into the third cerebral ventricle of conscious, unrestrained male rats, outfitted with chronic third ventricle and jugular cannulae, decreased the resting prolactin (PRL) levels. NT (5 micrograms) also markedly inhibited the PRL-releasing effect of activation of central serotonin receptors by 5-hydroxytryptophan (5HTP, 15 mg/kg) in animals pretreated with fluoxetine (4 mg/kg). however, interruption of dopamine (DA) neurotransmission by either alpha-methyl-p-tyrosine (alpha MT, 250 mg/kg) or spiroperidol (0.3 mg/kg) led to a blockade of the PRL-inhibiting effect of NT. Treatment of animal with alpha MT prior to the injection of fluoxetine and 5HTP also blocked the PRL-inhibiting effect of NT. These results suggest that the central dopaminergic system mediates the PRL-inhibitory effect of NT.

Maturational effect of triiodothyronine and its analog 3,5-dimethyl-3'-isopropyl-L-thyronine on sucrase activity in the small intestine of the developing rat.

In this study we compared the effect of 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT) with that of triiodothyronine (T3) on sucrase activity in the small intestine of the fetal and suckling rat. DIMIT (5 micrograms/100 g BW) was injected into pregnant Sprague--Dawley rats on days 17-20 of gestation and DIMIT (10 and 50 micrograms/100 g BW) and T3 (10 and 50 micrograms/100 g BW) were given subcutaneously to two groups of sucklings on days 4-7 and 11-14. Fetal animals were sacrificed on day 21 of gestation; sucklings on days 8 and 15. Sucrase activity in the proximal, middle, and distal small intestine was assayed. DIMIT and T3 suppressed serum thyroid-stimulating hormone (TSH) in the fetus and suckling. Sucrase activity was increased in all small intestinal segments in the fetal and older suckling groups. In the younger sucklings the effect was seen only in the middle and distal segments. In both fetal and suckling groups the sucrase response was dose related. Thus, DIMIT has a thyromimetic effect on development of sucrase activity. The lack of sucrase reactivity in the proximal segment of the younger (8-day-old) sucklings to the administration of either DIMIT or T3 is interesting but unexplained.

The effects of neurotensin on anterior pituitary hormone secretion.

The present experiments were conducted to determine the effects of neurotensin on secretion of a variety of anterior pituitary hormones. Conscious rats with indwelling cannulae in the third ventricle and external jugular vein were used and the effects on plasma hormone levels measured by radioimmunoassay. Neurotensin was found to decrease plasma prolactin levels in ovariectomized females, normal males, and males in which prolactin levels had been elevated by ether or by a combination of fluoxetine and 5-hydroxytryptaphane. The prolactin-lowering effect was blocked by alpha-methyl-tyrosine to inhibit catecholamine synthesis and by the specific dopamine receptor blocker, spiroperidol. In ovariectomized females, neurotensin was also capable of suppressing LH and elevating growth hormone following its intraventricular injection. Intravenous injection of the peptide elevated prolactin but had no effect on the release of the other pituitary hormones. When hemipituitaries of ovariectomized rats were incubated in vitro, neurotensin elevated prolactin and TSH release into the medium. The minimal effective dose to elevate prolactin and TSH release was 50 ng/ml. Release of gonadotropins and growth hormone was unaffected. It is concluded that neurotensin inhibits prolactin release by a CNS, presumably hypothalamic action, to stimulate the tuberoinfundibular dopaminergic neurons. The dopamine released then inhibits prolactin release either by a direct action on the pituitary or by release of another prolactin-inhibiting factor. In addition, the peptide has a direct prolactin-releasing action on the pituitary. Neurotensin can inhibit LH and stimulate growth hormone presumably by a hypothalamic action since there was no effect on the release of these pituitary hormones by glands incubated in vitro. Although the peptide had no effect on TSH release following its intraventricular injection, it stimulated prolactin release by pituitaries incubated in vitro. The physiological significance of these results is not yet established; however, the presence of the peptide in regions concerned with pituitary control suggests that it may play a physiological role.

Neurotransmitter control of thyrotropin secretion.

The central dopaminergic system seems to have an inhibitory influence on the secretion of thyrotropin (TSH) both in humans and rats. This is documented by observations that in humans, especially in subjects with primary hypothyroidism, the dopamine precursor l-Dopa, dopamine receptor agonist bromocryptine, and dopamine itself decrease plasma levels of TSH and in some instances inhibit TSH secretory response to thyrotropin-releasing hormone (TRH). Conversely blockade of dopamine receptors leads to an elevation of circulating TSH. It is probable the dopaminergic drugs act on the pituitary thyrotrops rather than on the release of hypothalamic TRH. Analogous results were obtained in rats. In contrast, the noradrenergic system, studied mainly in rats, has a stimulatory influence on the secretion of TSH. This view is mainly supported by findings that acute interruption of noradrenergic neurotransmission causes a decrease of serum TSH levels and blocks the cold-induced stimulation of TSH secretion. The role of the central serotonergic system remains undecided because some experimental findings indicate that it has an inhibitory influence but others indicate an opposite function. The remaining transmitter systems have not been studied extensively enough to allow definite conclusions about their roles in the regulation of TSH secretion.