Pituitary Portal Plasma Research Articles

Increase of Gonadotropin-Releasing Hormone Concentration in Pituitary Portal Blood after Substance P Administration in Male Rats

Substance P (SP) affects gonadotropin release from the anterior pituitary gland. In the present study we tested whether SP exerts this effect through GnRH release into pituitary portal blood in intact male rats (INT), orchidectomized rats with s.c. chronically implanted empty Silastic capsule (ORCX), testosterone capsule (ORCX + T), and 17β-estradiol capsule (ORCX + E₂). The pituitary glands were exposed by the transpharyngeal approach under urethane-chloralose anesthesia. Then, the stalk portal vessels were cut and three 30-min portal blood samples were collected. Each first sample of blood was treated as a control before 0.2 ml injection of normal saline, 5 µg, or 25 µg of SP in 0.2 ml of normal saline into the internal carotid artery. GnRH concentration in the purified portal plasma were measured by RIA. Injection of SP into the internal carotid artery caused a significant increase in GnRH concentration in pituitary portal plasma only in INT rats. The higher dose of SP markedly increased GnRH concentration in the 1st blood sample (p < 0.001) and in the 2nd blood sample GnRH concentration was lower but still significant higher than prior SP injection (p < 0.05). The lower dose of SP increased GnRH concentration later, only in the 2nd portal blood sample after intracarotid SP injection (p < 0.001). Injection of normal saline had no effect on GnRH concentration in pituitary portal blood in INT rats. In ORCX, ORCX testosterone- and estrogen-implanted rats portal plasma GnRH concentrations were not changed significantly after injection of both doses of SP. These results indicate that SP stimulates GnRH release into pituitary portal blood and the influence of SP on GnRH neurons depends on the levels of circulating gonadal steroid hormones.

Diurnal Variation in Luteinizing Hormone-Releasing Hormone and β-Endorphin Release in Pituitary Portal Plasma during the Rat Estrous Cycle1

In this study, we investigated the pattern of both LHRH and beta-endorphin (beta-EP) secretion from the median eminence of cyclic female rats during the estrous cycle by collecting pituitary portal blood for hormone measurements by RIA. On diestrus, proestrus, or estrus (0900 h or 1300 h), rats were hypophysectomized (in order to prevent contamination of portal beta-EP with pituitary beta-EP) and were given a 50-60-min resting period of postsurgical recovery prior to blood sampling. Between 1000 h and 1800 h of the same day, pituitary portal blood samples were collected at 10-min intervals from these animals under Saffan anesthesia. Hormone RIA data revealed that LHRH and beta-EP levels in the portal plasma ranged from 25 to 600 pg/ml and from 0.6 to 4.6 ng/ml, respectively, whereas no discernible amounts of these hormones were detected in the peripheral plasma. Analysis further revealed that secretion of both LHRH and beta-EP showed a diurnal rhythmicity and that both neuropeptides were secreted in a pulsatile manner on each day of the cycle, with most dramatic changes seen in female rats on proestrus. In proestrus rats, portal plasma levels of beta-EP were highest during 1000-1500 h and lower around 1600-1800 h. In contrast, morning and early afternoon values of LHRH were obviously higher in rats on proestrus than in animals on other days of the cycle, and an even greater rise in portal plasma levels of LHRH occurred in late afternoon on proestrus, when beta-EP values were lowest.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen augments hypothalamicβ-endorphin secretion and activates an inhibitoryβ-endorphin short-loop feedback system

The role of estrogen in the regulation of hypothalamicβ-endorphin hormone secretion is studied by determiningβ-endorphin concentration in pituitary portal plasma of ovariectomized rats in the presence or absence of this steroid and/or the opioid antagonist naloxone. Twenty-six hours following s.c. injection of 10 /µg estradiol benzoate (estrogen) or oil, rats anesthetized with Saffan (alphaxolone/alphadolone) underwent pituitary stalk exposure and hypophysectomy, after which pituitary portal blood was continuously collected and stored in 15 min aliquots from 1100-1400 h. At 1100 h, animals were given an initial bolus iv injection of naloxone or saline (naloxone, 2 mg/ kg, or saline, 0.1 ml) and then infused (iv) continuously with naloxone (2 mg/kg/h) or saline (0.8 ml/h) until 1400 h. Plasma samples were extracted and assayed by radioimmunoassay forβ-endorphin. Treatment with estrogen increased the meanβ-endorphin levels twofold as compared to oil-treated controls. Naloxone potentiated estrogen action ofβ-endorphin secretion, but did not affect basalβ-endorphin secretion. These results suggest that estrogen enhancedβ-endorphin secretion from the hypothalamus. Furthermore, the hypersecretion ofβ-endorphin induced by naloxone with, but not without, estrogen supports the existence of an estrogen-activated short-loop negative feedback mechanism regulatingβ-endorphin secretion.

A central negative feedback action of thyroid hormones on thyrotropin- releasing hormone secretion

Two experiments were conducted to test the hypothesis that thyroid hormones exert central negative feedback effects on the secretion of TRH from the hypothalamus in the ewe. In the first experiment, we examined the effects of thyroidectomy on the secretion of TRH and TSH. Thyroidectomy was followed by an unambiguous increase in TRH in pituitary portal plasma and TSH in the peripheral circulation. In the second experiment, we tested the effects of T4 replacement to thyroidectomized ewes. T4 replacement reversed the effects of thyroidectomy on TRH and TSH release. Of interest, TRH secretion in thyroidectomized ewes was continuously elevated during the collection, raising the possibility that TRH is secreted continuously, rather than exclusively in a strictly pulsatile manner indicative of phasic discharges synchronized among TRH neurosecretory elements. Collectively, these results suggest that thyroid hormones can act centrally to inhibit TRH (and thus TSH) release in the ewe, and they support the concept that at least part of the negative feedback action of thyroid hormones is exerted at the hypothalamic level.

A central negative feedback action of thyroid hormones on thyrotropin-releasing hormone secretion.

Two experiments were conducted to test the hypothesis that thyroid hormones exert central negative feedback effects on the secretion of TRH from the hypothalamus in the ewe. In the first experiment, we examined the effects of thyroidectomy on the secretion of TRH and TSH. Thyroidectomy was followed by an unambiguous increase in TRH in pituitary portal plasma and TSH in the peripheral circulation. In the second experiment, we tested the effects of T4 replacement to thyroidectomized ewes. T4 replacement reversed the effects of thyroidectomy on TRH and TSH release. Of interest, TRH secretion in thyroidectomized ewes was continuously elevated during the collection, raising the possibility that TRH is secreted continuously, rather than exclusively in a strictly pulsatile manner indicative of phasic discharges synchronized among TRH neurosecretory elements. Collectively, these results suggest that thyroid hormones can act centrally to inhibit TRH (and thus TSH) release in the ewe, and they support the concept that at least part of the negative feedback action of thyroid hormones is exerted at the hypothalamic level.

Pituitary portal plasma levels of oxytocin during the estrous cycle, lactation, and hyperprolactinemia.

The median eminence receives fibers from both parvocellular and magnocellular OT neurons in the hypothalamus. The OT neuronal terminal in the median eminence is secretory and is the major source of the neuropeptide in the blood of pituitary portal vessels. The OT secretion into pituitary portal plasma increases by ovarian steroids, PRL, and the suckling stimulus. The OT secretion into the blood of pituitary portal vessels changes in parallel with the altered secretion of PRL from the pituitary. Because of correlative association between pituitary portal plasma OT and systemic plasma PRL and much abundant evidence for a direct stimulatory action of OT on PRL release, we propose that the pituitary portal vascular system serves as the window for the central OT neurotransmission to the pituitary lactotropes.

Correlative Changes of the Gonadotropin-Releasing Hormone and Gonadotropin-Releasing-Hormone-Associated Peptide Immunoreactivities in the Pituitary Portal Plasma in Female Rats

The precursor protein of gonadotropin-releasing hormone (GnRH) contains a 56-amino acid peptide, known as GnRH-associated peptide (GAP), and GnRH. Both of these peptides are localized in the same neurons and coprocessed under varieties of physiological conditions. In the present study, we evaluated whether these two peptides are cosecreted into the pituitary portal blood in female rats under the conditions in which the secretion of hypothalamic GnRH and pituitary luteinizing hormone (LH) are known to be altered. The immunoreactivities of GAP-like peptide (IR-GAP-LI) and GnRH (IR-GnRH) in the portal plasma were 2- to 15-fold higher than those observed in peripheral plasma of female rats. In the pubertal females, the preovulatory LH surge which occurred in the afternoon of the day before vaginal opening (puberty) was found to coincide with surges of IR-GAP-LI and IR-GnRH in the pituitary portal plasma. The surges of IR-GAP-LI and IR-GnRH in portal plasma corresponded with a fall in the preoptic and hypothalamic contents of these peptides. In the adult rats, the levels of IR-GAP-LI and IR-GnRH in portal plasma and LH in peripheral plasma were significantly higher during the afternoon of proestrus than those in the afternoon of diestrus. Ovariectomy increased the portal plasma levels of IR-GAP-LI and GnRH and peripheral plasma levels of LH as compared to the level of these hormones in diestrous females. These results indicate that both GnRH and GAP-LI are cosecreted into pituitary portal blood and that changes in the endocrine environment similarly affect both GnRH and GAP secretion.

Glucocorticoids but not vasopressin or oxytocin inhibit luteinizing hormone secretion in patients with psychogenic amenorrhea.

Activation of the hypothalamus-pituitary-adrenal (HPA) axis is suggested to play a role in the stress-related inhibition of LH secretion. The aim of our study was to investigate the effects of vasopressin and oxytocin, which are increased in pituitary portal plasma in response to stress, and of glucocorticoids, the final product of HPA activation during stress, on basal plasma LH levels and on pituitary LH response to the GnRH test in amenorrheic (n = 33) and fertile (n = 13) women. Plasma LH levels were evaluated by radioimmunoassay in 2 different experimental conditions: 1. Basal secretion; 2. The GnRH test (10 micrograms + 10 micrograms after a 120-minute interval). These 2 evaluations were done in the presence of both placebo and a pharmacological dose of desmopressin (an analogue of vasopressin) (16.6 ngr/minute), oxytocin (0.2 ngr/minute) or hydrocortisone (4.1 mg/minute). None of these drugs modified basal plasma LH levels either in amenorrheic patients or in controls. Hydrocortisone inhibited the GnRH-induced LH increase in amenorrheic women. These data suggest that the glucocorticoids might play a role in LH secretion and indicate a possible participation of the HPA axis in the impairment of the hypothalamus-pituitary-gonadal axis in women with psychogenic amenorrhea.

Evidence for Prolactin Feedback Actions on Hypothalamic Oxytocin, Vasoactive Intestinal Peptide and Dopamine Secretion

Ovariectomized rats, when transplanted with 4 anterior pituitaries (APs) to the kidney capsule for 2-3 weeks, had elevated plasma prolactin (PRL) levels (3.8-fold) and showed decreased in situ AP weights (0.62-fold) and PRL concentrations (0.63-fold). The concentrations of dopamine (DA) and oxytocin (OT) in pituitary portal plasma of hyperprolactinemic rats were increased 1.7- and 1.9-fold, respectively. However, the levels of vasoactive intestinal peptide (VIP) in pituitary portal plasma of these rats were decreased 0.31-fold. The secretion of DA, dihydroxyphenylalanine (DOPA) and OT from fetal hypothalamic cells in primary culture was increased, whereas VIP secretion from these cells was reduced in a dose-dependent fashion following PRL treatment. These data are the first in vivo and in vitro demonstration of a stimulatory action of PRL on OT release and an inhibitory action of PRL on VIP release. Furthermore, these data suggest that a subtle imbalance between the secretion of the PRL-inhibiting factor (DA) and the PRL-releasing factors (VIP and OT) during elevated systemic levels of PRL is responsible for decreased lactotrophic function.

High Concentrations of Neuropeptide Y in Pituitary Portal Blood of Rats

Pituitary portal blood was collected from urethane-anesthetized rats and examined for the presence of neuropeptide Y (NPY) using high-performance liquid chromatography and radioimmunoassay. Other rats were perfused with fixative, and coronal sections through the hypothalamus and median eminence were processed for immunohistochemical localization of NPY. Combined high-performance liquid chromatography and radioimmunoassay analysis of pituitary portal plasma and systemic plasma revealed a single peak of NPY immunoreactivity which corresponded in retention time to synthetic porcine NPY. Increasing amounts of portal or systemic plasma produced displacement curves which were parallel to the NPY standard curve. The concentration of NPY immunoreactivity in portal plasma (52.0 +/- 4.0 ng/ml, mean +/- SEM) was three times greater (p less than 0.005) than in systemic plasma (16 +/- 4.5 ng/ml). NPY-labeled fibers were observed in the external zone of the median eminence in the vicinity of hypothalamo-hypophyseal portal vessels. The observation of significantly higher concentrations of NPY immunoreactivity in the portal plasma supports the hypothesis that NPY may be released from the hypothalamus to affect pituitary function.

Hyperprolactinemia decreases the luteinizing hormone-releasing hormone concentration in pituitary portal plasma: a possible role for beta-endorphin as a mediator.

Hyperprolactinemia can reduce the LH secretion in rats, but the mechanism of the effect of PRL is not clear. We have investigated the actions of PRL on the secretion of LHRH and LH and the interaction among PRL, beta-endorphin (beta-EP), and LHRH. The effects of PRL on LHRH and LH secretion were studied in ovariectomized female rats after transplanting four anterior pituitaries to the right kidney capsule of each ovariectomized rat for 2-3 weeks. The level of PRL in rats with pituitary transplants was approximately 5 times higher than that in control rats. The concentration of LHRH in pituitary portal plasma of hyperprolactinemic rats was approximately 4 times lower than that in control rats. Hyperprolactinemic animals also showed lower plasma LH levels than the controls. Since beta-EP inhibits the secretion of LHRH, we have tested whether the reduced secretion of LHRH in hyperprolactinemic ovariectomized rats is associated with an increase in beta-EP activity. This was studied by measuring the concentration of beta-EP in pituitary portal plasma and the response of LHRH and LH to the opiate antagonist naloxone. The level of beta-EP-like immunoreactivity in pituitary portal plasma was significantly higher in hyperprolactinemic rats than in control animals. Naloxone (10 mg/kg, sc) increased both LHRH and LH concentrations in hyperprolactinemic rats, but not in control rats. The present results demonstrate that hyperprolactinemia can reduce LHRH release and suggest a possible involvement of beta-EP in the PRL inhibitory action on LHRH.

Inhibition of gonadotropin-releasing hormone release as the basis of pituitary-gonadal dysfunction during treatment with 4-aminopyrazolo-(3,4-d)-pyrimidine.

The inhibitor of hepatic lipoprotein release, 4-aminopyrazolo-(3,4-d)-pyrimidine (4-APP), has been shown to reduce testosterone production via impairment of pituitary gonadotropin secretion rather than through decreased cholesterol availability. It was previously shown that serum LH levels were reduced by more than 75% in male rats treated with 4-APP, but pituitary stores of LH and the gonadotropin response to exogenous GnRH were maintained. Also, there was a reduction in pituitary GnRH receptors which was consistent with hypothalamic GnRH deficiency. The present studies were undertaken to examine the mechanism by which 4-APP inhibits GnRH synthesis and/or release. Intact, adult male or 2-week ovariectomized female rats were treated daily with 25 mg/kg 4-APP for 3 days. Both sexes showed lowered basal serum levels of LH and absence of the elevations in serum LH normally elicited by the opiate antagonist, naloxone. In pituitary portal plasma collected from normal male rats, GnRH was significantly elevated by naloxone treatment, confirming that naloxone acted at the level of hypothalamic GnRH release. However, naloxone stimulation of GnRH secretion into portal blood was absent in rats treated with 4-APP. In vitro, the potassium-induced release of GnRH from perifused medial hypothalami was reduced by 60% in 4-APP-treated male rats while hypothalamic GnRH content remained unchanged. These data indicate that 4-APP has an inhibitory effect on the mechanism of GnRH release, and that analysis of its actions should clarify the processes involved in neurohormone secretion.

Studies on the central and peripheral dopaminergic control of PRL secretion in man: failure to demonstrate the existence of a dopamine-mediated prolactin-inhibiting factor.

The existence of a putative central prolactin-inhibiting factor (PIF) distinct from dopamine (DA) but dependent on DA mechanisms for release has been suggested from recent animal studies. We investigated the possibility of the existence of such a PIF in man by combining the use of monoiodotyrosine (MIT), an inhibitor of central DA synthesis, domperidone, a DA receptor antagonist that does not enter the blood-brain barrier and DA itself. 6 normal volunteers underwent three sets of studies: (1) PRL stimulation test to 400 micrograms TRH i.v., 1 g MIT orally or 5 mg domperidone i.v., (2) peripheral DA receptor blockade study in which either domperidone, MIT or TRH was administered at 120 min during a 240-min domperidone infusion (50 micrograms/min) which was preceded by a 5-mg bolus dose of domperidone i.v. and, (3) DA infusion study in which MIT was administered at 120 min during a 240-min infusion of DA in a dose (0.5 microgram/kg X min) known to elevate peripheral DA concentration to levels reported for pituitary portal plasma. In the PRL stimulation tests, the mean +/- SE peak response was significantly greater (p less than 0.002) with domperidone (3,900 +/- 840 mIU/l) than with MIT (1,880 +/- 400 mIU/l) or TRH (2,094 +/- 450 mIU/l). In the peripheral DA receptor blockade study the initial domperidone-induced PRL response was not sustained during the domperidone infusion. Neither a second dose of domperidone nor MIT administration at 120 min resulted in a significant release of PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic variation of oxytocin in the blood of pituitary portal vessels of rats.

The release of oxytocin (OT) into pituitary portal blood during the estrous cycle was studied in female rats under Althesin anesthesia. On each afternoon of the 4-day estrous cycle, animals were anesthetized with Althesin, and pituitary portal blood samples were collected. The concentration of OT in portal plasma collected between 14.00 and 16.00 h of proestrus (1,079 +/- 185 pg/ml) was significantly higher than that at any other time studied. There were no appreciable differences between the mean OT concentration on the days of estrus (198 +/- 29 pg/ml), metestrus (283 +/- 55 pg/ml), and diestrus (267 +/- 49 pg/ml). The concentration of OT in pituitary portal plasma was also greater than that in blood withdrawn from the external jugular vein (less than 41-67 pg/ml). Comparison of the portal plasma OT with peripheral plasma prolactin in Althesin-anesthetized rats indicated an association between the release of these hormones during the estrous cycle.

Dopaminergic control of gonadotrophin secretion in normal women and in patients with pathological hyperprolactinaemia.

The role of dopaminergic mechanisms in the control of gonadotrophin secretion in normal and hyperprolactinaemic subjects is controversial. Whilst bromocriptine, a potent dopamine agonist, has been used to restore normal gonadotrophin secretion in subjects with pathological hyperprolactinaemia (PHP), dopamine and dopamine agonists have been reported to suppress basal and stimulated gonadotrophin release. We therefore investigated the importance of dopaminergic control of gonadotrophin secretion by studying LH, FSH and PRL responses in normal and PHP subjects to central dopamine synthesis inhibition using monoiodotyrosine (MIT) and to a 4 h dopamine infusion designed to elevate peripheral plasma dopamine concentration to levels reported for pituitary portal plasma (1-6 ng/ml). MIT administration resulted in a significant release of PRL (peak increment 520 +/- 84% above basal) but not of LH or FSH in normal subjects. In PHP subjects there was a blunted PRL response (peak 13.3 +/- 3.5%) to MIT administration and significant LH (P less than 0.05) but not FSH release. Dopamine infusion (0.5 microgram/kg/min) resulted in suppression of PRL (min 19 +/- 3% of basal) but not of LH or FSH. A rebound of PRL (peak 188 +/- 68% of basal) but not LH or FSH occurred on cessation of dopamine. There was an apparent rise in LH (P less than 0.02 vs. normals) but not FSH in PHP patients during dopamine infusion. Plateau dopamine levels achieved during the infusion were 2.9 +/- 0.3 ng/ml and 5.9 +/- 0.8 ng/ml in normal and PHP subjects respectively. The responses to MIT show that dopamine functions as an inhibitor of PRL but not of LH or FSH in normal subjects. In PHP patients the responses suggest increased dopaminergic inhibition of LH release but loss of inhibitory control of PRL release. Physiological concentrations of plasma dopamine do not significantly inhibit LH or FSH release in normal subjects but paradoxically results in an apparent release of LH in PHP patients. We conclude that dopamine mechanisms do not play a significant role in modulating gonadotrophin release in normal subjects. In PHP patients, PRL feedback results in increased hypothalamic dopamine activity which in turn inhibits LH release. We conclude that the inhibitory action of dopamine on PRL release restores LH secretion by removing central dopaminergic inhibition through hypothalamic feedback of PRL.

Dopamine infusion studies in patients with pathological hyperprolactinemia: evidence of normal prolactin suppressibility but abnormal dopamine metabolism.

The cause of pathological hyperprolactinemia (PHP) is not known, although it has been postulated that lactotroph refractoriness to dopamine (DA) is an important factor. We, therefore, studied PRL suppressibility in response to iv DA administered in a dose designed to achieve peripheral plasma DA concentrations in the range reported in rat pituitary portal plasma. Nine normal subjects and 14 patients (8 with probable microadenomas and 6 with macroadenomas) with PHP were studied during a 4-h infusion of 0.5 micrograms/kg X min DA. Serum PRL levels decreased in normal subjects to 18 +/- 3% (mean +/- SEM) of initial basal levels, not significantly different from the fall to 22 +/- 2% of basal levels found in patients with PHP. Maximal PRL suppression in patients with probable microadenomas (21 +/- 2%) was not significantly different from that in those with macroadenomas (22.8 +/- 2.0%). Upon cessation of DA infusion, there was a rapid rebound in PRL release, which was significantly greater (P less than 0.05) in patients (155 +/- 15%) than in normal subjects (118 +/- 13%). Plasma DA levels were measured by gas chromatography/mass spectroscopy and were significantly higher (P less than 0.05) in patients than in normal subjects during the infusion. The MCR for DA in normal subjects (12.5 +/- 2.8 liters/kg X h) was significantly greater (P less than 0.005) than that in patients (6.2 +/- 0.5 liters/kg X h). The data show that the release of PRL in patients with PHP is not refractory to physiological concentrations of DA, but that there is an abnormality of peripheral DA metabolism in these patients. We conclude that the development of PHP is unlikely to be due to insensitivity of lactotrophs to endogenous DA and that patients with PHP have a systemic disturbance of DA metabolism.

Morphine suppresses the proestrous surge of GnrH in pituitary portal plasma of rats.

Virgin proestrous rats were acutely treated with 2 doses of morphine sulfate (MS) (5-8 and 40 mg/kg BW) or naloxone HCl (NH) (10 mg/kg BW) co-administered with the high dose of MS, and the pituitary portal plasma concentrations of gonadotropin releasing hormone (GnRH) were compared with those of untreated proestrous (PE) and diestrous (DE) control animals. LH and FSH were measured in systemic plasma obtained by venipuncture just prior to the collection of portal blood. Both doses of MS severely diminished the PE surges of LH and FSH, but NH reversed the effect of MS and restored the circulating gonadotropins to PE levels. However, only PE rats treated with the high dose of MS exhibited significantly reduced GnRH concentrations in portal plasma. This suggests that the reduction of gonadotropin concentrations is not due merely to reduced GnRH secretion by the hypothalamus but may involve other mechanism(s) as well. However, in rats given the high dose of MS the severe reduction in pituitary gonadotropin secretion is attributable in large part to the corresponding decrease in hypothalamic GnRH release, since NH restored the GnRH and LH/FSH plasma concentrations to PE levels.

Synthetic corticotropin-releasing factor stimulates secretion of immunoreactive β-endorphin/β-lipotropin and ACTH by human fetal pituitaries in vitro

Synthetic ovine CRF, in an amount approximating that found in pituitary portal plasma of the rat, induced a significant increase in the secretion of both ACTH and immunoreactive β-endorphin/β-LPH (iβ-END/LPH) by human fetal hemipituitaries in an in vitro superfusion system. This finding suggests that a molecule similar to synthetic ovine CRF may be a physiologic hypothalamic releasing factor in man.