Absence of short-loop autoregulation of prolactin during late pregnancy in the rat.

Animals bearing intrahypothalamic anterior pituitary (AP) grafts exhibit a central hyperprolactinemia, and thus, serve as a model for the study of short-loop feedback regulation of prolactin (PRL) secretion. We investigated the effects of intrahypothalamic AP grafts on PRL secretion during late pregnancy (n = 7). A further group of five rats was injected with the dopamine against bromocriptine during late pregnancy and five control rats were treated with the bromocriptine-vehicle only. A nocturnal surge in plasma PRL concentrations was observed in the vehicle-injected control animals, peaking at 212 +/- 11 ng/ml at 0300 h on the day of parturition. Despite the central hyperprolactinemia due to the grafts, a similar PRL surge was observed in grafted animals, peaking at 205 +/- 35 ng/ml at 0300 h on the day of parturition. Bromocriptine treatment completely blocked the nocturnal surge of PRL. These results suggest that short-loop feedback autoregulation of PRL secretion becomes less responsive or nonfunctional in the last 24 h of pregnancy in the rat. This apparent change in sensitivity of the autofeedback mechanism may be an important physiological mechanism to allow the hypersecretion of PRL during lactation.

Effect of alcohol, neurohypophysectomy, and vasopressin antagonists on hemorrhage-induced bradycardia in the rat.

During initial stages of hemorrhage in the rat, cardiovascular compensation leads to a tachycardia (mean +/- SE, 5.2 +/- 0.7%; n = 23) that helps prevent a large fall in blood pressure. This compensatory phase is followed by a decompensatory phase in which mean arterial pressure and heart rate fall. A rise in arginine vasopressin (AVP) levels has been postulated as the cause of this hemorrhage-induced bradycardia (HIB). The object of the present study was to determine whether interference with AVP release by alcohol anesthesia or neurohypophysectomy or by blockade of AVP receptors in the plasma or cerebral spinal fluid could attenuate HIB. Male Wistar rats were anesthetized with pentobarbital, surgically prepared, and bled to maintain a blood pressure of 40-50 mm Hg. After hemorrhage, heart rate decreased 15 +/- 2% (n = 6) with alcohol anesthesia compared with 32 +/- 3% (n = 7) with pentobarbital. After neurohypophysectomy, however, HIB remained unchanged (-15 +/- 2%; n = 5) compared with sham-operated controls (-19 +/- 3%; n = 6). Peripheral administration of two nonselective V1/V2 antagonists and one V2 antagonist had no effect on HIB, whereas a V1 antagonist significantly attenuated the heart rate decrease (-15 +/- 4%; n = 6) compared with controls (-32 +/- 3%; n = 7). None of the AVP antagonists tested at one tenth the peripheral dose had any effect on HIB when administered into the lateral ventricle of the brain, although a mixed serotonin, dopamine, and catecholamine antagonist, spiperone, potentiated the response. It was concluded that although peripheral release of AVP may be partially involved in the heart rate response to hemorrhage, central AVP release and central AVP receptors were not involved in HIB.

Neurohormonal Factors Involved in the Control of the Nocturnal Prolactin Surge that Precedes Parturition in the Rat.

A nocturnal surge of prolactin (PRL) occurs in the dark period preceding parturition in the rat. The roles of oxytocin, vasoactive intestinal peptide (VIP), serotonin and the opioids in controlling the antepartum PRL surge were investigated by examining PRL secretion over the last 2 days of pregnancy in the presence of antagonists to these neurohormonal factors. Serial blood samples were collected from unanesthetized, freely moving rats via indwelling jugular cannulae, and plasma PRL was measured by radioimmunoassay. In control rats PRL levels rose in a nocturnal surge peaking at 223 ± 34 ng/ml (n = 6) at 0500 h on day 21 of pregnancy, the day of parturition. Intra-arterial infusion of the oxytocin antagonist desGly-NH(2) d(CH(2) )(5) [Tyr(Me)(2) , Thr(4) ]-OVT at a dose sufficient to completely block milk ejection (10 µg/h) had no effect on this PRL surge. Infusion of the VIP antagonist [4Cl-D-Phe(6) ,Leu(17) ]-VIP at 2 µg/h from 2200 h on day 20 until 0500 h on day 21 significantly attenuated the antepartum PRL surge, reducing the peak to 76 ± 28 ng/ml at 0500 h on day 21 (n = 6; P<0.001). Naloxone, the opiate receptor antagonist, inhibited the antepartum PRL surge in a dose-dependent manner. Infused at 2 mg/h naloxone partially reduced the magnitude of the PRL surge, which peaked at 128 ± 24 ng/ml at 0300 h on day 21 (n = 4; P<0.05), while at 10 mg/h naloxone totally abolished the PRL surge (n = 6; P<0.001). Injection of the serotonin synthesis inhibitor ρ-chlorophenylalanine (250 mg/kg, sc at 1700 h on days 19 and 20 of pregnancy) increased the magnitude of the antepartum PRL surge to a peak of 327 ± 48 ng/ml at 0500 h on day 21 (n = 5), compared with 244 ± 24 ng/ml at the same time in vehicle-injected controls (P<0.05; n = 5). The results demonstrate that the antepartum PRL surge is stimulated by an opioid mechanism, and also by VIP. Oxytocin and serotonin have no role in stimulating PRL secretion during late pregnancy.

Role of the placenta in the control of the ante-partum surge of prolactin in the rat.

A nocturnal surge of prolactin secretion occurs in the dark period preceding parturition in the rat. The aim of this study was to examine the role of the placenta in the control of this prolactin surge. Plasma prolactin and progesterone were measured by radioimmunoassay in serial blood samples collected after surgical removal of conceptuses during late pregnancy, and after intracerebroventricular (i.c.v.) injection of placental lactogen (PL) before the prolactin surge. In intact control animals, prolactin secretion remained low until a nocturnal surge of secretion occurred in the dark period preceding parturition, peaking at 269 +/- 51 (S.E.M.) micrograms/l at 03.00 h on day 21. Progesterone levels fell from greater than 200 nmol/l on day 19 to less than 40 nmol/l by 12.00 h on day 20 of pregnancy. PL levels during late pregnancy were modified by partial or complete removal of conceptuses at 10.00 h on day 19 of pregnancy. Removal of all but one or two conceptuses did not change the normal pattern of prolactin or progesterone secretion. Removal of all conceptuses, however, induced a large nocturnal surge of prolactin secretion, peaking at 211.7 +/- 78 micrograms/l at 03.00 h on day 20, 24 h earlier than the surge in intact animals. Progesterone levels after removal of all conceptuses fell to less than 40 nmol/l by 23.00 h on day 19, approximately 12 h before the decline in intact animals. Maintenance of increased progesterone levels after conceptus removal using silicone tubing implants significantly (P less than 0.05) reduced the peak of the premature prolactin surge to 79.7 +/- 18 micrograms/l at 05.00 h on day 20.(ABSTRACT TRUNCATED AT 250 WORDS)

Posterior pituitary lobectomy chronically attenuates the nocturnal surge of prolactin in early pregnancy.

The posterior pituitary gland contains a potent PRL-releasing factor (PRF). The aim of this study was to determine whether this PRF was involved in the luteotropic PRL surges of early pregnancy. The posterior pituitary was removed from a group of rats at least 4 weeks before the experiment (LOBEX). LOBEX rats were tested for diabetes insipidus and checked postmortem for the absence of posterior lobes. Plasma samples were taken from these chronically LOBEX rats as well as from sham-operated and unoperated controls beginning at noon on day 3 of pregnancy and continuing for 3 days at times most likely to demonstrate the twice daily surges of PRL secretion. PRL in all groups was basal (less than 12 ng/ml) at noon and rose to a diurnal peak at 1800 h. While the unoperated and sham-operated controls then displayed a clear nocturnal surge of PRL, peaking at 300-400 ng/ml at 0300 h, LOBEX rats had a significantly attenuated peak of 102.7 +/- 17.7 ng/ml (P less than 0.001). On subsequent nights the nocturnal peaks were similarly attenuated in LOBEX rats, averaging 80.3 and 42.1 ng/ml on days 5 and 6, respectively. LOBEX rats all had normal sized litters, but lactation yields were depressed. These data support the hypothesis that the posterior pituitary secretes a potent PRL-releasing factor because its removal partially blocked the mating-induced nocturnal surge of PRL.

Intrahypothalamic pituitary grafts elevate prolactin in the cerebrospinal fluid and attenuate prolactin release following ether stress.

Anterior pituitary (AP) tissue grafted into the hypothalamus of female rats inhibits the luteotrophic prolactin (PRL) secretion which normally follows mating. Dopamine blockade has been shown to overcome this inhibition, suggesting that the grafts suppress PRL release from the in situ pituitary by the action of graft PRL increasing dopamine activity in the hypothalamus. To examine whether PRL levels in the cerebrospinal fluid (CSF) were elevated by the AP grafts, CSF samples were taken from 5 control rats and 10 rats bearing intrahypothalamic AP grafts. Mean PRL concentrations in the CSF of the control rats were 3.0 +/- 0.8 ng/ml. The grafted rats had significantly higher concentrations of PRL in their CSF, averaging 23.2 +/- 4.2 ng/ml (P less than 0.005). Plasma PRL concentrations were similar in the control and grafted rats. PRL release in response to 5 min of ether stress was examined in 8 control and 11 grafted rats. In control animals, PRL rose from 4.2 +/- 1.5 to 44.7 +/- 9.0 ng/ml following exposure to ether, but the response was significantly attenuated in the grafted rats, peaking at 9.3 +/- 1.4 ng/ml (P less than 0.001). This inhibition of response due to the grafts was evident within 1 week of graft placement. The results confirm that the presence of intrahypothalamic AP grafts led to the accumulation of supranormal PRL concentrations in the CSF. This elevated PRL suppressed pituitary PRL release in response to ether stress, probably by an autoregulatory feedback activation of the inhibitory tuberoinfundibular dopaminergic neurons in the hypothalamus.

Effect of ovarian steroids on a nocturnal surge of prolactin secretion that precedes parturition in the rat.

PRL secretion in several physiological and experimental conditions, including early pregnancy, is linked to the daily photoperiod. The aim of this study was to examine the antepartum increase in PRL secretion for evidence of a circadian pattern of release, as seen during early pregnancy. During the last 3 days of pregnancy blood samples were taken six times daily by means of previously implanted jugular cannulae. Plasma PRL concentrations were then measured by RIA. PRL levels were less than 10 ng/ml in all animals on day 19 of pregnancy, but during the light period of day 20 there was an increase to an average of 30 +/- 10 ng/ml, with no evidence of a peak related to the time of day. However, in the dark period between days 20 and 21 there was a large surge of PRL secretion which reached peak levels of 356 +/- 39 ng/ml at 0500 h on day 21, then returned to 48 +/- 20 ng/ml at 1200 h, around the time of parturition. The peak always occurred at 0500 h and was not related to the time of parturition which ranged from 1000-2200 h on day 21. Bilateral ovariectomy (OVX) on day 19 advanced the time of delivery by approximately 12 h. In seven of nine animals, no surge of PRL secretion was observed during the dark period preceding parturition. Estradiol treatment after OVX on day 19 (OVX+E) advanced the time of delivery by approx 18 h. An antepartum PRL surge was present and was advanced by 24 h in all OVX+E animals, peaking at 0300 h on day 20. Progesterone treatment from day 18 to 21 in intact pregnant animals delayed parturition by approximately 18 h and prevented PRL secretion during the period of treatment. After progesterone treatment was stopped, a nocturnal surge of PRL secretion occurred, peaking at 0500 h on day 22, 24 h after the surge in normal animals. The results suggest that the increased PRL secretion during late pregnancy is linked to the daily photoperiod and is characterized by a nocturnal surge in the dark period preceding parturition. This surge is inhibited by progesterone, and it can be advanced 24 h by estradiol treatment in the absence of the ovaries.

Failure of an indirect competitive enzyme-linked immunosorbent assay to measure prolactin in rat plasma samples.

An indirect competitive enzyme-linked immunosorbent assay (ELISA) for rat prolactin was applied to the measurement of prolactin (PRL) in plasma. The assay involved PRL adsorbed to a 96-well plate competing with soluble PRL in the sample for rabbit anti-rat PRL antibody-binding sites. Antibody bound by the PRL on the well was detected by a goat anti-rabbit immunoglobulin antibody conjugated to the enzyme horseradish peroxidase. Dilutions of PRL in plasma-free assay buffer produced accurate and reliable standard curves over a range of 1-500 ng/ml. Plasma or serum samples, however, gave very low absorbance values, suggesting falsely high levels of PRL in the samples. For example, hypophysectomized rat plasma and fetal calf serum gave readings equivalent to over 250 ng/ml rat PRL by ELISA, whereas radioimmunoassay confirmed the expected PRL values of less than 5 ng/ml. This result indicated considerable nonspecific interference by the plasma. Inclusion of 20% or 50% hypophysectomized rat plasma in the assay significantly lowered the absorbance values and the slope of the standard curve (P less than 0.05). Dilution of plasma samples 1:1 and 1:4 with assay buffer caused nonlinear changes in absorbance and resulted in inaccurate ELISA estimates of plasma PRL concentrations compared with radioimmunoassay. Several treatments were attempted to remove the interference from plasma. Preexposure of plasma to 56 degrees C for 30 min, dialysis for 24 hr, fractional precipitation with 5% polyethylene glycol, increased ionic strength of the buffer, preadsorption of plasma with rabbit immunoglobulins bound to a solid phase support, and covalent attachment of the PRL to the wells with 2% glutaraldehyde all failed to remove the interference from the plasma samples in the ELISA. Thus, while the assay worked effectively for plasma-free applications, it was unsuitable for measurement of PRL in plasma or serum samples.

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.

The inhibition of prolactin secretion due to intrahypothalamic pituitary grafts is reversed by estradiol benzoate but not by progesterone.

Anterior pituitary (AP) tissue grafted into the hypothalamus inhibited the luteotrophic response to mating and prevented pseudopregnancy (PSP) and pregnancy. All normal rats given 10 micrograms estradiol benzoate (EB) on estrus became PSP (15 days) while the same treatment caused 10-day PSPs in 20/21 grafted rats. Doses of 30 micrograms EB or 10 micrograms EB plus reserpine (1 mg/kg) resulted in 15-day PSP in grafted rats. By contrast progesterone (P; 10 mg on estrus) did not prolong cycles in rats with hypothalamic grafts though it did in 50% of normals. Earlier studies showed that PSP or pregnancy was restored in the grafted rats by blockade of dopamine (DA) secretion. The results above show that EB was similarly effective in restoring PSP while P was not, suggesting that EB both raised prolactin and lowered DA while P was unable to lower DA in rats with AP grafts in the hypothalamus.

Dopamine blockade reverses the inhibition of prolactin which results from intrahypothalamic pituitary grafts.

When mated with fertile bucks, rats with anterior pituitary (AP) tissue grafted into the hypothalamus did not exhibit prolongation of the diestrous cycle. Treatment of these rats with alpha-methyl-p-tyrosine, reserpine, or haloperidol for 1 or 2 days after mating increased the interestrous interval by a few days in all rats and to more than 8 days (leading to pseudopregnancy or pregnancy) in 20% of the cases. The same treatment in unmated normals resulted in 80% becoming pseudopregnant. To get more than 70% of rats with hypothalamic AP grafts pregnant or pseudopregnant required dopamine-blocking drugs for 3 or 4 consecutive days. Pregnancy was prolonged in 50% and lactation was impaired in 78% of the grafted rats which littered. Both impairments, like the original failure of the luteotrophic response, are attributed to the effects of PRL autofeedback from the hypothalamic AP grafts. These experiments provide further evidence that the mechanism whereby PRL in the hypothalamus inhibits PRL secretion involves elevation of dopamine.