Biomedical subjects

M E Freeman

Publications and source records attributed to M E Freeman.

At least 55 records · Page 3Linked to original sources

PubMed1993

Infusion of dopamine at low concentrations stimulates the release of prolactin from alpha-methyl-p-tyrosine-treated rats.

Prolactin (PRL) secretion from the anterior pituitary gland is inhibited by dopamine (DA) released into the hypophysial portal vasculature from neurons in the hypothalamus. We have shown previously that DA also stimulates PRL secretion in vitro. Here we report that DA has a dual effect on PRL release in vivo. Injection of rats with alpha-methyl-p-tyrosine (200 mg/kg, ip) induced an immediate 35-fold enhancement of PRL secretion which reached a plateau by 90 min after injection on diestrus 1. When DA was infused intravenously at varying doses beginning at 90 min after alpha-methyl-p-tyrosine, differing effects on PRL secretion were observed. These effects were dose dependent: higher doses of DA (1000 ng/kg/min) inhibited and lower doses (10 ng/kg/min) stimulated PRL secretion. These data suggest that DA may be an important stimulator of PRL secretion in vivo.

Animals↗

PubMed1992

Activity of oxytocinergic neurons in the paraventricular nucleus mirrors the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion.

PRL secretion is regulated by an endogenous stimulatory rhythm of PRL-releasing factors within the hypothalamus. The endogenous rhythm has a bimodal periodicity with a nocturnal component which peaks at approximately 0300 h and a diurnal component that peaks at approximately 1700 h. Several PRL-releasing factors are known to be involved in this rhythm. Among these are oxytocin (OT), vasoactive intestinal peptide, and serotonin. We have proposed that OT is the neurohormone that stimulates PRL release from the lactotroph. In this study, we examined the activity of OTergic neurons in the paraventricular nucleus using the expression of the protooncogene c-fos (Fos) as a marker of neuronal activity. Ovariectomized rats were killed at either 0300, 1200, or 1700 h and brains quickly fixed by perfusion with 2.5% acrolein in 4% paraformaldehyde. Brains were blocked and processed for OT/Fos immunohistochemistry. Rats killed at 0300 and 1700 h had significantly greater proportion of Fos expressing OTergic neurons than control rats (1200 h). Percent of Fos-positive OTergic neurons were 2- and 1.5-fold greater at 0300 and 1700 h than 1200 h, respectively. The majority of these neurons were located in the medial parvocellular paraventricular nucleus and periventricular area. In another experiment, groups of OVX rats were killed every 2 h over a 24-h period and OT extracted from their anterior and posterior pituitaries. OT was present in the anterior pituitary in a bimodal rhythm. OT concentrations were greatest at approximately 0400 h and slowly declined to baseline by 1000 h. Another peak of OT was present in the anterior pituitary at approximately 2000 h and quickly declined to baseline by 2400 h. This rhythm of OT was not reflected in either the posterior pituitary or trunk blood. These data suggest that activity of a specific population of OTergic neurons of the paraventricular nucleus is rhythmic. The periodicity of these neurons mirrors that of the endogenous stimulatory rhythm. Furthermore, the anatomical location of these neurons suggests that they may project to the median eminence. Indeed, this heightened activity is reflected in a bimodal rhythm of OT in the anterior pituitary. Taken together, the data presented here provide compelling support for the role of OT as the neurohormone in the mechanism of the endogenous stimulatory rhythm.

Animals↗

PubMed1992

The stimulatory and inhibitory effects of dopamine on prolactin secretion involve different G-proteins.

The reverse hemolytic plaque assay (RHPA) was used in this study to further characterize the mechanism whereby low concentrations of dopamine (DA) stimulate PRL secretion in vitro. Female Sprague-Dawley rats were used as a source of anterior pituitary cells for the RHPA. Pituitary cells were infused into Cunningham chambers along with a suspension of protein-A-coated ovine red blood cells. Excess cells were rinsed from the chambers leaving a monolayer of cells attached to the glass. The cells were then incubated with solutions containing PRL antiserum (1:40) and various concentrations of DA. After 4 h, a solution containing guinea pig complement (1:60) was infused into the chambers. Thirty minutes later, the cells were fixed and plaques (zones of hemolysis) surrounding PRL-producing cells (lactotrophs) were measured and used as an index of the amount of PRL secreted. Control cells that received no DA had a mean plaque area of 8,000 microns 2 and two distinct subpopulations of plaque sizes. This biphasic population of cells consisted of a small and a large plaque producing population. The mean plaque area surrounding lactotrophs was significantly (P less than 0.05) decreased if 1 microM or 10 microM DA was present (4,500 microns 2 and 3,500 microns 2, respectively). These cells which received inhibitory concentrations of DA demonstrated a monophasic distribution of plaque-forming cells. On the other hand, mean plaque area was significantly (P less than 0.05) increased if 0.1 nM or 1 nM DA was presented to the cells (15,000 microns 2 and 14,500 microns 2, respectively). These cells receiving stimulatory doses of DA exhibited a multiphasic distribution of plaque-forming cells. The possibility that the two physiological opposing actions of DA on PRL secretion might be mediated by different GTP binding proteins was also examined using cholera toxin (CTX) and pertussis toxin (PTX). Anterior pituitary cells were pretreated with either CTX (50 micrograms/ml) or PTX (5 micrograms/ml) for 1 h before initiation of the RHPA. In the RHPA, cells received no DA, a stimulatory dose of DA (0.1 nM), or a inhibitory dose of DA (10 microM). The effects of toxin pretreatment on mean plaque area of DA-treated cells was determined. PTX pretreatment significantly attenuated the inhibitory effects of DA while having no effect on the stimulatory effects of DA on PRL secretion. CTX significantly (P less than 0.05) potentiated the stimulatory effects of DA on PRL secretion and had no effect on inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Activity of vasoactive intestinal peptide and serotonin in the paraventricular nucleus reflects the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion.

PRL secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR) of prolactin-releasing factors of hypothalamic origin which has a bimodal periodicity with distinct nocturnal (N) and diurnal (D) phases. The N phase reaches peak magnitude by 0300 h and the D phase reaches peak magnitude by 1700 h. This rhythm was first unmasked in ovariectomized rats by correctly timed injection of a dopamine antagonist. OT, vasoactive intestinal peptide (VIP), and serotonin (5-HT) are differentially involved in generating the ESR. Pharmacological studies suggest that OT is the neurohormone and VIP and 5-HT are neuromodulators which act to stimulate OT release. Recently, we reported that activity of OTergic neurons in the paraventricular nucleus (PVN) and OT concentrations in the anterior pituitary mirror the periodicity of the ESR. The present experiments were conducted to determine if VIP and 5-HT activity in the hypothalamus also mirrors the periodicity of the ESR. Push-pull cannulae were surgically implanted in the PVN of ovariectomized female rats. Following recovery, push-pull perfusion was conducted from either 0600-1400 h, 1400-2200 h, or 2200-0600 h. VIP was measured in perfusates by RIA. There was no difference in VIP pulse frequency between rats perfused during the three periods studied. However, animals perfused from 2200-0600 h had significantly greater pulse amplitude as compared to rats at either 0600-1400 h or 1400-2200 h. Activity of 5-HTergic neurons in the hypothalamus was studied by estimating the turnover of 5-HT 10 min following the injection of pargyline. Hypothalamic nuclei were dissected using Palkovits' punch technique and 5-HT concentration assayed by HPLC in conjunction with electrochemical detection. Turnover of 5-HT was estimated by calculating the slope of the accumulation of 5-HT over 10 min at differing times of day using least squares regression analysis. There was a distinct diurnal rhythm of 5-HT accumulation in the PVN. Rats killed at 1700 h had significantly greater slopes of 5-HT accumulation in the PVN than rats killed at either 0300 or 1200 h. Similarly, there was a diurnal rhythm of 5-HT turnover in the suprachiasmatic nucleus. Rats sampled at either 1200 or 1700 h had significantly greater slopes of 5-HT accumulation in the suprachiasmatic nucleus than rats sampled at 0300 h. There was no diurnal rhythm of 5-HT turnover evident in either the median eminence or the supraoptic nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Inhibition of prolactin secretion by endothelin-3 is pertussis toxin-sensitive.

The effect of pertussis toxin (PTX) pretreatment on endothelin-3 (ET-3)-mediated inhibition of prolactin secretion from primary cultures of rat anterior pituitary cells was examined. Monolayer cultures of anterior pituitary cells were treated with either 20 ng/ml PTX dissolved in media or with media alone (control) on the third day of culture. Exactly 24 h after PTX pretreatment, cells were challenged with either 100 nM ET-3 dissolved in media or media alone (control) for 4 or 48 h. ET-3 significantly (P less than 0.01) inhibited prolactin secretion in both the 4 and 48 h incubations. However, if the cells had been previously treated with PTX, ET-3 did not significantly affect prolactin secretion. These data suggest that a PTX-sensitive G protein mediates ET-3-induced inhibition of prolactin secretion and that ET-3 may invoke a signal transduction mechanism in the lactotroph which is distinct from those described in other cell types.

Animals↗

PubMed1991

Endothelin-3 inhibits prolactin and stimulates LH, FSH and TSH secretion from pituitary cell culture.

The influence of endothelin-3 (ET-3) on anterior pituitary hormone secretion was investigated over a wide range of concentrations (from 10(-14) to 10(-6) M) and incubation times (from 4 to 48 hours). ET-3 elicited a concentration-dependent inhibition of prolactin (PRL) secretion and stimulated the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH) from primary monolayer cultures of anterior pituitary cells derived from female rats. The responsiveness of different pituitary cells to ET-3 differs markedly in terms of onset and duration: the maximal inhibition of PRL secretion occurred after 12 hours and the stimulation of LH, FSH and TSH reached the maximum after 4, 48 and 48 hours of incubation, respectively. These data corroborate the concept that ET-3 has an important role as a neuroendocrine modulator. Moreover, the data presented suggest different intracellular mechanisms underlying ET-3 actions.

Animals↗

PubMed1991

The endogenous stimulatory rhythm regulating prolactin secretion is present in the lactating rat.

Prolactin (PRL) secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR), which is normally under the tonic inhibition of dopamine (DA). The ESR consists of a nocturnal (N) component which peaks at approximately 03.00 h, and a diurnal (D) component which peaks at approximately 17.00 h. This ESR has been shown to be present in ovariectomized and cervically stimulated rats. We have proposed that the ESR is continuously present in the female rat and that any suppression of the tonic inhibitory influence on PRL secretion can reveal its existence. In this study, the effects of the DA-lowering stimulus of suckling was investigated at different times of day in lactating rats. In addition, the pattern of PRL secretion in freely lactating rats throughout a 24-hour period was studied. Female rats were separated from their pups for 6 h prior to reunion at either 03.00 (coincident with the N component), 12.00 (control) or 17.00 h (coincident with the D component) at various stages of lactation. Blood samples were collected from intra-atrial cannulae immediately before separation of pups and dams, immediately before reunion of pups and dams (0 time), and 15, 30, 60 and 120 min following reunion of pups and dams. Four days following parturition, dams suckled at either 03.00 or 17.00 h secreted significantly greater PRL than rats suckled at 12.00 h. Peak levels of PRL were 60-, 90- and 25-fold greater than 0 time levels, at 03.00, 17.00 and 12.00 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Pharmacologic evidence that a D2 receptor subtype mediates dopaminergic stimulation of prolactin secretion from the anterior pituitary gland.

The ability of low concentrations of dopamine (DA) to stimulate the secretion of prolactin (PRL) was examined in perifused or monolayer cultures of anterior pituitary cells. In cultures perifused with media containing 100 nM DA, changing the DA concentration to either 1 or 100 pM caused a significant dose-dependent stimulatory PRL secretory response within 6 min when compared to the PRL secretory response to removal of DA altogether. Picomolar concentrations of DA caused a biphasic PRL secretory response. This response is characterized by an immediate increase in the rate of PRL secretion similar to that seen when the cells were treated with 100 nM thyrotropin-releasing hormone followed by a decrease in the rate of PRL secretion to levels comparable to cells receiving media alone. In a monolayer culture system DA, at concentrations between 10 nM and 1.0 pM, caused significant stimulation of PRL secretion relative to media alone. Maximal stimulation occurred at nanomolar concentrations of DA (approximately 60% greater than control). Although the D2 agonists, bromocriptine and 2-(N-phenethyl-N-propyl)-amino-5-hydroxytetralin hydrochloride (PPHT) caused significant (p less than 0.05) inhibition of PRL secretion at nanomolar concentrations and above, neither had stimulatory activity. The D1 agonists, SKF 38393 and SKF 82958, had no effect on PRL secretion when tested at 0.1 pM to 1 microM. These data suggest that DA not only inhibits PRL secretion in vitro, but also stimulates PRL secretion at relatively low concentrations. Stimulation is mediated by a DA receptor which is neither recognized by D2 nor D1 agonists, suggesting a possible third DA receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Ontogeny of the endogenous stimulatory rhythm regulating prolactin secretion in immature female rats.

PRL secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR). This rhythm consists of two components: a nocturnal (N) component whose activity is greatest by 0300 h and a diurnal (D) component that peaks at approximately 1700 h. This periodicity coincides with the periods of the N and D surges of PRL in responses to the mating stimulus. Furthermore, we have shown that the ESR is involved in the regulation of mating-induced PRL surges. Mating causes a lowering of dopaminergic tone which reveals the ESR for PRL. The ability of immature female rats to express PRL surges induced by copulomimetic stimuli begins at 25 days of age. In this study we investigated the ontogeny of the ESR in immature female rats in order to observe the relationship between the onset of PRL secretion induced by copulomimetic stimuli and the development of the ESR. Immature female rats were raised in our colony and kept with their dams until used in an experiment or weaned at 23 days of age where appropriate. At 15, 20, 23, 24, 25, or 30 days of age female rats received a single ip injection of domperidone (DOM; 5 mg/kg) or saline vehicle at 0300, 1200, or 1700 h. Thirty minutes after the injection the rats were decapitated, and trunk blood was collected. PRL was measured by RIA. DOM had no effect on PRL secretion as compared to that in saline-treated controls at 15 days of age. However, in all other age groups DOM induced a significant increase in PRL levels compared to those in saline-treated animals regardless of the time of injection. In addition, there was no time of day difference in the PRL secretory response to DOM in rats 15-23 days of age. However, rats treated with DOM at 0300 h at 24 days of age secreted approximately 2-fold greater PRL than rats treated similarly at 1200 or 1700 h. Moreover, at 25 and 30 days of age, rats treated with DOM at either 0300 or 1700 h secreted significantly greater PRL than rats treated similarly at 1200 h. These results suggest that the ESR for PRL secretion begins by 24 days of age. In addition, they indicate that the hypothalamic developmental event preceding and required for expression of mating-induced surges of PRL is the establishment of the ESR.

Aging↗

PubMed1991

The effects of endothelins on the secretion of prolactin, luteinizing hormone, and follicle-stimulating hormone are mediated by different guanine nucleotide-binding proteins.

Different bacterial toxins capable of modifying specific alpha-subunits of G-proteins were used to characterize the guanine nucleotide-binding protein (G-protein) dependency of the effects of endothelins (ETs) on PRL, LH, and FSH secretion. Primary cultures of anterior pituitary cells obtained from female rats were preincubated for 24 h with 20 ng/ml pertussis toxin (PTX) or 2 micrograms/ml cholera toxin (CTX) before challenge with ETs. Both ET-1 and ET-3 elicited a concentration-dependent inhibition of PRL secretion and stimulated the release of LH and FSH secretion on pituitary cells not treated with toxins. Based on the calculated ration of the half-maximal effective concentrations (EC50) of ET-1 and ET-3, ET-1 showed 7800, 20, and 14 times greater potency than ET-3 on PRL, LH, and FSH secretion, respectively. PTX, a selective inhibitor of Gi and several other G proteins, increased the basal secretion of PRL and completely eliminated the responsiveness of lactotroph cells to ET-1 and ET-3. Pretreatment with PTX caused a markedly different effect on LH and FSH secretion: while basal LH release was slightly increased, FSH secretion was markedly depressed by PTX. Moreover, while ET-induced LH secretion was enhanced by PTX, the effectiveness of ETs on FSH release was completely abolished. CTX, known as an activator of Gs proteins, decreased the basal secretory activity of lactotrophs but did not influence the ET-induced decrease of PRL release. CTX pretreatment (like PTX before) elicited a strikingly different effect on LH and FSH: while basal LH secretion was enhanced, basal FSH secretion was markedly inhibited by CTX. Moreover, while the effectiveness of ETs on LH secretion was not changed significantly, the stimulatory effect of ETs on FSH secretion was diminished after CTX pretreatment. Thus, the inhibition of PRL secretion by ETs requires a PTX-sensitive G protein while the ET-induced stimulation of FSH secretion involves both PTX- and CTX-sensitive elements. The fact that pretreatments with PTX or CTX influenced basal secretion of PRL, LH, and FSH suggests that PTX- and/or CTX-sensitive G proteins are directly involved in the process of exocytosis. Additionally, these findings might indicate an active paracrine/autocrine regulation of pituitary cells in culture that are impaired or enhanced by the bacterial toxins employed. Though the broad substrate specificity of PTX and CTX and the multiplicity of G protein families did not allow us to identify the specific G protein(s) involved, these data reveal the diversity of ET-induced intracellular signaling mechanisms in lactotrophs and gonadotrophs.

Animals↗

PubMed1990

Oxytocin, vasoactive-intestinal peptide, and serotonin regulate the mating-induced surges of prolactin secretion in the rat.

In female rats the mating stimulus induces a bimodal pattern of PRL secretion. A surge of PRL occurs at approximately 0300 h, called the nocturnal surge (N). Another surge occurs at 1700 h on the same day, called the diurnal surge (D). By lowering dopaminergic tone pharmacologically, we have recently demonstrated the existence of an endogenous rhythm stimulatory to PRL secretion in female rats. The periods of this stimulatory influence coincide with the periods of the N and D surges of PRL that occur in mated rats. In addition, we have shown that the 0300 h component of this endogenous rhythm is regulated by oxytocin (OT) and vasoactive intestinal peptide (VIP), and the 1700 h component is regulated by OT and serotonin (5-HT). In this study, we investigated the roles of OT, VIP, and 5-HT in controlling the N and D surges of PRL in ovariectomized (OVX) rats receiving a physiological dopamine-lowering stimulus, copulomimetic stimulation. Blood samples were obtained the day before the experiments between 1700 and 1900 h to verify that the rats used were having surges of PRL in response to cervical stimulation (CS). The role of OT was studied by infusing the OT antagonist 1-deamino-2-D-Trp4-Val8-Orn-OT (OT-A; 0.5 micrograms/kg.min) beginning at either 0100 or 1500 h and continuing for 5 h on day 2 after the last CS. Serial blood samples were obtained immediately before infusion and 60, 90, 120, 150, 180, 240, and 300 min after the start of infusion. The samples overlapped either the N or D surge of PRL. All rats used in these studies demonstrated D surges of PRL the day before the experiment. Saline infusion had no effect on either the N or D surge of PRL in OVX-CS rats. However, infusion of OT-A completely blocked both the N and D surges of PRL. The role of VIP was studied by infusing the VIP antagonist [4-D-Cl-Phe6-Leu17]VIP (VIP-A; 01 micrograms/kg.min) beginning at either 0100 or 1500 h and continuing for 5 h. VIP-A completely blocked both the N and D surges of PRL. To study the role of 5-HT, rats received an acute treatment with p-chlorophenylalanine (PCPA; 250 mg/kg, sc) at either 0100 or 1500 h, and blood samples were taken as before. PCPA had no effect on the N surge of PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Hysterectomy-induced alterations in prolactin secretion of lactating rats.

The contribution of the uterus to the regulation of PRL secretion in lactating dams and cycling female rats was investigated. Lactating animals were hysterectomized or sham operated 2 days after parturition, and the number of pups was adjusted to eight. Blood samples for PRL RIA were obtained through intra-atrial cannulae implanted 2 days before experimentation. In order to study the PRL secretory profile in undisturbed freely lactating rats, blood samples were taken every 2 h for 24 h starting at 1400 h. During early lactation (days 7-8), hysterectomy did not alter the PRL secretory profile compared to that of sham-operated controls. On days 14-15 post partum, PRL secretion followed a characteristic bimodal pattern showing two PRL surges at 1800 h and 0600 h. After hysterectomy, the early morning PRL surge disappeared and PRL secretion showed an unimodal daily rhythm reaching its peak at 1800 h. The possible effect of the absence of the uterus on suckling-induced PRL release at various stages of lactation was studied. On days 7-8, suckling stimuli after 4 h of pup deprivation induced robust PRL release. Hysterectomy did not significantly alter PRL release at this earlier stage of lactation. In control groups, the suckling-induced PRL secretory response markedly declined as the postpartum period advanced. On the other hand, the hysterectomized animals retained significantly greater responsiveness to suckling during the second half of lactation. These data indicate an inhibitory influence of the uterus on PRL secretion. The onset of this uterine effect is considerably delayed, and its influence became prominent only at a later phase of lactation. The effect of length of pup deprivation preceding the suckling stimulus, in combination with hysterectomy, was also investigated. Hysterectomy significantly increased suckling-induced PRL release after 4 and 24 h separation compared to the sham-hysterectomized animals. When the separation was longer than 48 h, the inducibility of PRL release by suckling declined and was not influenced by hysterectomy. In order to study the possible influence of the uterus on PRL secretion during the estrous cycle, regularly cycling female rats were hysterectomized at diestrus 1. Twelve days later the animals were cannulated, and serial blood samples were taken during the subsequent proestrus. Hysterectomy did not alter the PRL surge which occurred on the afternoon of proestrus indicating that the uterus does not have a major function in regulating PRL secretion on proestrus. In conclusion, hysterectomy significantly delayed the extinction of suckling-induced PRL release revealing the active role of the uterus in the regulation of this neuroendocrine reflex.

Animals↗

PubMed1989

A sex-specific endogenous stimulatory rhythm regulating prolactin secretion.

In the rat, PRL secretion is under inhibitory control by tuberoinfundibular dopaminergic neurons. The levels of dopamine (DA) in hypophysial portal blood decline during surges of PRL secretion (e.g. suckling and cervical stimulation). However, this decline alone is not sufficient to account for the amount of PRL released. In this study we investigated the possible existence of an endogenous stimulatory rhythm for PRL secretion that may be masked by the tonic inhibitory tone of DA and unmasked by the DA-lowering effects of cervical stimulation. The PRL secretory response to pharmacological depression of DA-ergic tone was studied in ovariectomized (OVX) female, adult castrated (AC) male, neonatally androgen-sterilized (TP) female, and neonatally castrated (NC) male rats. Since mated rats have serum PRL surges at 0300 and 1700 h, these groups were treated with 200 micrograms/kg domperidone (DOM), iv, at 0300 h, 1700 h, or the intersurge interval, 1200 h. Serial blood samples were collected immediately before and at frequent intervals after DOM injection. OVX female rats had significantly greater serum PRL responses to DOM at 0300 and 1700 h than at 1200 h. AC male rats secreted significantly less PRL in response to DOM compared to OVX rats, and their PRL responses to DOM were similar at all three times. TP female rats had PRL secretory responses similar to those of the OVX rats at 1200 h, and the responses at 0300 and 1700 h were similar. NC male rats had PRL secretory responses similar to those of AC male rats. There was no difference between the PRL secretory profiles at any time after DOM injection in NC rats. These data provide evidence for an endogenous stimulatory rhythm for PRL secretion that is specific to female rats. They further suggest that the neonatal steroid environment is critical for differentiation of some sexually specific characteristics.

Animals↗

PubMed1989

Hypothalamic factors involved in the endogenous stimulatory rhythm regulating prolactin secretion.

We recently reported that acute pharmacologic depression of dopaminergic tone at different times of day unmasks a sex-specific endogenous stimulatory rhythm regulating PRL secretion. The PRL secretory responses of ovariectomized rats to the dopamine antagonist domperidone (DOM) were higher at 0300 and 1700 h than at 1200 h. These are the times during which surges of PRL appear in mated rats. This experimental paradigm was used to investigate the roles of the putative PRL-releasing factors (PRFs) oxytocin (OT), vasoactive intestinal peptide (VIP), and serotonin (5-HT) in this rhythm. The role of OT was studied by infusion of the OT antagonist 1-deamino-2-D-Trp-4-Val-8-Orn-Oxytocin (OT-A, 0.5 microgram/kg min) for 6 h. Two hours after beginning the OT-A infusion DOM was administered, as a single injection of 200 micrograms/kg iv at either 0300, 1200, or 1700 h. Serial blood samples were collected immediately before and 5, 10, 20, 30, 60, 120, 180, and 240 min after DOM administration. Infusion of OT-A attenuated the heightened PRL secretory responses to DOM given at both 0300 and 1700 h but did not affect the response at 1200 h. The role of VIP was studied by infusing the VIP antagonist [D, 4-Cl-Phe6,Leu17] VIP (VIP-A, 0.1 microgram/kg.min) as described above. VIP-A infusion had no effect on the PRL secretory responses to DOM given at 1200 or 1700 h but attenuated the heightened response at 0300 h. In order to study the role of 5-HT in the rhythm, rats were pretreated with p-chlorophenylalanine (250 mg/kg sc) 48 and again 24 h before the experiment. Pretreatment with p-chlorophenylalanine had no effect on the PRL secretory responses to DOM given at 0300 or 1200 h, but it attenuated the augmented PRL secretory response at 1700 h. These data suggest that both VIP and OT act as endogenous PRFs at 0300 h and 5-HT and OT act as PRFs at 1700 h. We propose that VIP and 5-HT are continuously active oscillatory neurotransmitters regulating OT release into pituitary portal blood and that these daily events only eventuate in PRL release when the mating stimulus has release the lactotroph from the inhibitory effects of dopamine.

Animals↗

PubMed1987

Partial purification of a hypothalamic factor that inhibits gonadotropin-releasing hormone-stimulated luteinizing hormone release.

A protein that suppresses GnRH-stimulated LH release from dispersed rat anterior pituitary cells was purified from rat hypothalami by chromatography on Sephadex G-25, carboxymethyl cellulose, and high performance gel permeation. The final increase in inhibitory activity was 214-fold. The inhibitor is a glycoprotein with an apparent mol wt of 12,252 +/- 638, as determined by gel permeation on HPLC and electrophoreses as a monomer after treatment with 8 M urea. The inhibitor has a Stokes radius of 16 A, an S value of 2.14 +/- 0.08, an isoelectric point value near 4.1, and a frictional ratio of 1.05. A preliminary amino acid composition is presented.

Animals↗

PubMed1987

A physiological role for luteinizing hormone release-inhibiting factor of hypothalamic origin.

A 12 KD peptide, LH release-inhibiting factor (LHRIF), extracted from rat hypothalamus has been shown to inhibit LHRH-stimulated LH release from cultured anterior pituitary cells. In the present study this partially purified material also blocks LHRH-stimulated LH release in pentobarbital-blocked proestrous rats. Administration of a polyclonal antiserum to partially purified LHRIF potentiates estrogen-induced LH release and antagonizes the inhibitory effect of estrogen and progesterone on LH release in ovariectomized rats. These results suggest that a hypothalamic LHRIF plays a physiological role in regulating LH release in the rat.

Animals↗

PubMed1986

Membrane currents in identified lactotrophs of rat anterior pituitary.

Qualitative features of the primary inward and outward current components of identified lactotrophs of the rat anterior pituitary were examined. Identification of lactotrophs in heterogeneous dissociated anterior pituitary cultures was accomplished by application of the reverse hemolytic plaque assay. Currents in lactotrophs were subsequently examined using whole-cell or patch recording techniques. Two components of inward calcium current were observed: a transient component and a sustained component. The transient component activated at voltages as negative as -50 mV and was the major contributor to total lactotroph calcium current. The sustained component activated at voltages above about -10 mV. The 2 currents could be qualitatively separated by differences in inactivation properties and in sensitivity to cadmium. At least 3 components of outward current were distinguished. Either 30 mM TEA or 0 calcium eliminated a major portion of sustained outward current. This is likely to represent primarily calcium- and voltage-activated potassium current. The remaining current could be further differentiated into a transient current component that could be inactivated with conditioning potentials above -60 mV. A slowly activating and deactivating potassium current remained following inactivation of the transient current. Although the time course of the transient current is reminiscent of "A" current, activation of this current required potentials above -30 mV. Candidates for the single-channel currents that underlie the whole-cell outward currents were observed in cell-attached recordings. When combined with patch-clamp electrophysiological methods, the reverse hemolytic plaque assay promises to be a powerful technique for the electrophysiological characterization of specific cell subtypes in heterogeneous dissociated cell populations.

Animals↗

PubMed1985

Effects of placenta and maternal serum on prolactin secretion in vitro.

The effects of the placenta and maternal sera on the secretion of prolactin (Prl) were examined in vitro. Placentae were obtained on each of Days 8-11 of pregnancy and extracted in 2.0% butanol-saline. To determine if these extracts could inhibit Prl secretion in vitro, dispersed anterior pituitary cells were incubated with placental extracts containing 1.0 placental equivalent obtained on each of Days 8-11 of pregnancy. Prl secretion was not affected by extracts of placentae obtained on Day 8 but was significantly inhibited by placental extracts obtained on Days 9-11 of pregnancy. In fact, progressively more mature placentae induced greater degrees of Prl inhibition. Extracts of placentae that were obtained on each of Days 8-11 of pregnancy, normalized on the basis of protein and tested for a 24-h period in the dispersed pituitary bioassay, caused the same degree of inhibition over Prl release. Additionally, placental protein from any given day (Days 8-11) of pregnancy induced a highly significant dose-dependent inhibition over Prl secretion. Equivalent amounts of a nonspecific protein, bovine serum albumin, had no effect. These findings indicate that the placenta does indeed contain a Prl inhibitory factor whose specific activity remains relatively constant between Days 8 and 11 of pregnancy. To determine if the inhibitory activity is humoral, maternal sera collected on each of Days 8-11 of pregnancy were placed in culture with dispersed pituitary cells at a concentration of 15.0%. Concomitant with gestational maturity, there was a progressively greater inhibition of Prl release. These findings indicate that the placenta may secrete a substance into the blood which suppresses Prl release directly at the level of the pituitary gland.

Animals↗