The addition of charcoals to broiler diets did not alter the recovery of Salmonella Typhimurium during grow-out.

Two experiments evaluated prebiotics added to feed on the recovery of Salmonella in broilers during grow-out and processing. In Experiment 1, "seeder" chicks were inoculated with Salmonella Typhimurium and placed with penmates. Treatments were: basal control diet, added 0.3% bamboo charcoal, 0.6% bamboo charcoal, or 0.12% Aromabiotic (medium chain fatty acids). The ceca from seeders and penmates were sampled to confirm Salmonella colonization at 3, 4, and 6 wk, and pen litter was sampled weekly. At 3 wk, charcoal fed chicks had significantly lower cecal recovery (37% lower) of Salmonella via direct plating but no differences at wk 4 or 6. At 6 wk, broilers fed Aromabiotic had no recovery of Salmonella from ceca with direct plating and significantly, 18%, lower recovery with enrichment. In Experiment 2, the treatments were: basal control diet, added 0.3% bamboo charcoal, 0.3% activated bamboo charcoal, or 0.3% pine charcoal. At placement, 2 seeders were challenged with Salmonella and commingled with penmates and ceca sampled at 1 and 2 wk, and ceca from 5 penmates/pen at 3 to 6 wk. Weekly, the pH of the crop and duodenum was measured from 1 penmate/pen and the litter surface sampled. At the end of grow-out broilers were processed. Results showed that penmates had colonized at 1 and 2 wk. Cecal Salmonella showed no differences except at 4 wk, when activated bamboo charcoal had a 18% lower recovery of Salmonella (enrichment) compared to the control (88%). Similar to Experiment 1, the recovery of Salmonella from the litter was not significantly different among treatments, however an overall decrease in recovery by 4 wk with direct plating reoccurred. The pH of the duodenum and the crop were not different among treatments. Crop pH (6.0) for all treatments were significantly higher at wk 1 compared to wk 2 to 6. Charcoals had minimal effect on Salmonella recovery in the ceca, but following defeathering, broilers fed charcoals had significantly lower Salmonella recovery from breast skin (charcoals 5+/60 compared to control 8+/20). While the addition of charcoals to broilers feed did not significantly affect Salmonella recovery during production (from litter or ceca samples) there was a lower Salmonella recovery from breast skin following scalding and defeathering.

The influence of a twice-a-day feeding regimen after photostimulation on the reproductive performance of broiler breeder hens.

Broiler breeder hens are typically provided a restricted amount of feed once a day. This feed is rapidly consumed; therefore, the hens fast for an extended period of time before their next feeding. In the current research, the effects on reproductive performance of implementing a twice-a-day vs. a once-a-day feeding program after photostimulation were investigated. Pullets and cockerels were reared on a skip-a-day feeding program. Pullets were weighed at 20 wk of age and then distributed into 30 laying pens such that each pen had a similar BW distribution. Each individual laying pen consisted of 35 hens and 4 roosters. At 21 wk of age, the birds were photostimulated for reproduction and 15 of the laying pens were placed on a once-a-day feeding schedule, whereas the other 15 pens were placed on a twice-a-day feeding schedule. The total amount of feed provided per day to all the laying pens was the same. Birds fed once a day received all their feed at 0630 h, whereas birds fed twice a day received 60% of their total feed allotment at 0630 h and the other 40% at 1500 h. Even though both treatment groups began egg production at the end of wk 23, birds fed twice a day laid more (P < or = 0.05) eggs through 42 wk of age than those fed once a day. Additionally, the average egg weight for the entire production period, which lasted until the birds were 60 wk of age, was greater for hens fed twice a day. Overall BW uniformity for the entire experiment was significantly better for hens fed twice a day vs. once a day. However, cumulative mortality was significantly higher for hens fed twice a day than for those fed once a day. The results indicate that feeding broiler breeder hens twice a day after photostimulation may enhance reproductive performance during the early lay period.

Dietary nitrogen intake regulates hepatic malic enzyme messenger ribonucleic acid expression.

Increased dietary protein intake rapidly (3 h) decreases malic enzyme and increases hepatic histidase mRNA expression. Experiments were conducted to determine the role that individual dispensable amino acids and nonprotein N sources might have in regulating the activity of these enzymes and to determine if the addition of a N supplement to a practical broiler diet during the entire rearing period would reduce abdominal fat accumulation in broilers. Broiler chicks were fed a basal diet containing 22% protein or this diet supplemented with 9.5% l-Glu, 5% Gly, 6% l-Ala, 5.08% ammonium bicarbonate, or 4.25% dibasic ammonium phosphate for 24 h. Each of the dietary supplements added 0.90% total N to the diet. Hepatic malic enzyme mRNA expression was significantly (P < 0.05) depressed in chicks fed any of the supplemented diets compared with chicks fed the basal diet. Histidase mRNA expression, however, was only significantly increased in the chicks fed the basal diet supplemented with Gly. Broilers fed practical corn-soybean meal starter and developer diets supplemented with 2.3, 4.7, or 9.5% Glu from 0 to 40 d of age had significantly smaller abdominal fat pads relative to BW than broilers fed the unsupplemented corn-soybean meal diets. Feeding the Glu supplements, however, reduced the overall BW gain of broilers by 100 to 150 g compared with broilers fed the unsupplemented diets. The results suggest that hepatic mRNA expression of malic enzyme may be regulated by total dietary N intake, whereas hepatic mRNA expression of histidase may be regulated by specific amino acids.

The genetic and molecular organization of the Dopa decarboxylase gene cluster of Drosophila melanogaster.

We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.

Hypothalamic sites which control the surges of prolactin secretion induced by cervical stimulation.

Artificial stimulation of the uterine cervix (CS) of the rat institutes two daily surges of PRL. These surges in turn activate and maintain luteal progesterone secretion for a 13-day interval known as pseudopregnancy. Electrolytic lesions of the medial preoptic area (MPOA) or electrical stimulation of the dorsomedial-ventromedial areas (DMN-VMN) of the hypothalamus also induce pseudopregnancies. This study was designed to determine if these pseudopregnancies are also characterized by the biphasic pattern of PRL secretion. Bilateral electrolytic lesions were placed in the MPOA on the morning of either estrus or the 14th day after ovariectomy. Both experimental models had nocturnal (0500 h) but no diurnal (1900 h) surges of PRL on the second day after placement of the lesion. Sham-lesioned rats had neither surge. When both surges were initiated by CS on proestrus, placement of the lesion on estrus blocked the diurnal but not the nocturnal surge of PRL. These data suggest that the MPOA possesses neurons which are inhibitory to nocturnal surges and stimulatory to diurnal surges of PRL and that CS removes the inhibition over the former and stimulates the latter. Electrical stimulation of the DMN-VMN initiates both surges of PRL in intact or ovariectomized rats. Thus, CS may also act either directly or indirectly w;thin the DMN-VMN to induce both surges of PRL.

Detection of prolactin inhibitory activity in uterine epithelial cell secretions and rat serum.

We have shown that the uterus of the rat contains a substance that diminishes the release of the luteotropic hormone PRL by acting directly at the anterior pituitary gland. This study was designed to determine which cell type(s) within the uterus secretes this PRL inhibitory activity (PIA) and if PIA of uterine origin appears in circulation. Enzymatically dispersed cells from uteri of ovariectomized (OVX) rats were cultured for 24 h in serum-free Dulbecco's Modified Eagle's Medium. Estimation of PIA in the spent media from heterogeneous uterine cell cultures was evaluated after 24 h by the ability to suppress PRL release from confluent monolayers of cultured pituitary cells. Spent media from uterine cell cultures containing 0.125 X 10(6) or 0.25 X 10(6) cells/well failed to significantly suppress PRL secretion. However, media from 0.5 X 10(6) and 1 X 10(6) uterine cells/well induced 36% and 85% inhibition of PRL release, respectively, without affecting basal LH release. To determine which cell type(s) in the uterus is responsible for PIA secretion, whole uteri were partitioned into epithelial, stromal, and myometrial cell fractions by differential enzymatic dissociation. Varying numbers of cells from each fraction were cultured for 24 h. PIA in the spent media from the homogeneous uterine cell cultures was estimated by its ability to suppress PRL release from cultured anterior pituitary cells. Media from epithelial cell cultures suppressed PRL secretion in a dose-dependent fashion. Media obtained from cultures of a comparable number of stromal or myometrial cells had no significant effects on PRL secretion. LH secretion was unaffected by media obtained from any concentration of the various uterine cell types. Since cultured anterior pituitary cells treated directly with crude uterine extract for 24 h recover and secrete PRL at the same rate as untreated controls during a 72-h posttreatment interval, it is unlikely that the uterine PIA is either proteolytic or cytotoxic. To determine if PIA is detectable in peripheral circulation, sera obtained from OVX or OVX hysterectomized (OVX-HYST) donor rats were incubated with cultured anterior pituitary cells for 24 h. In response to the OVX sera, there was significant inhibition of PRL release. However, upon replacement of OVX sera with OVX-HYST sera, the inhibition was significantly reduced. Thus, PIA is higher in sera of rats bearing uteri. Taken together, these data suggest that the epithelial layer of the uterus secretes a PIA which probably reaches the hypothalamo-pituitary axis through the circulation.

Low concentrations of dopamine increase cytosolic calcium in lactotrophs.

Dopamine (DA) plays a dual role in the neuroendocrine regulation of PRL secretion from the anterior pituitary gland. DA normally exerts a tonic inhibitory effect on PRL secretion. However, DA at concentrations much lower than those required for maximal inhibition of PRL secretion actually stimulates PRL secretion. In this study, the effects of stimulatory concentrations of DA on common second messenger systems were examined in pituitary cells enriched for lactotrophs in an attempt to identify the signal transduction mechanisms used in DAergic stimulation of PRL secretion. Rat pituitary cells were enriched for lactotrophs (88% lactotrophs) by differential sedimentation on a discontinuous Percoll gradient. Enriched cells in monolayer culture were responsive to both inhibitory and stimulatory doses of DA; 10 microM DA inhibited PRL secretion by 80%, and 0.1 nM DA stimulated PRL secretion by 40% after a 4-h incubation. No dose of DA (10 pM to 10 microM) had an effect on phosphatidylinositol turnover. However, 1 and 10 microM DA inhibited cAMP formation by 58% and 72%, respectively. DA at doses in the stimulatory range had no effect on cAMP formation. Single cell cytosolic calcium concentrations ([Ca2+]i) were examined by quantitative fluorescence microscopy using fura-2 as a probe. Inhibitory doses of DA (1 microM) decreased [Ca2+]i in 83% of the cells examined. Two subpopulations of cells were noted that varied in the degree of response to 1 microM DA. One subpopulation responded to DA by decreasing [Ca2+]i 50-100 nM. The other subpopulation responded to DA by decreasing [Ca2+]i 300-400 nM. A stimulatory dose of DA (0.1 nM) increased [Ca2+]i in 44% of the cells examined. This dose of DA increased [Ca2+]i by 100-400 nM. These data indicate that the second messenger mediating DAergic stimulation of PRL secretion is most likely Ca2+.

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.

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)

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.

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.

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)

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.

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.

The imprint provided by cervical stimulation for the initiation and maintenance of daily prolactin surges: modulation by the uterus and ovaries.

This study describes the influence of ovarian steroids and a putative PRL release inhibitory substance of uterine origin on the imprint provided by cervical stimulation (CS) for the maintenance of nocturnal and diurnal surges of PRL secretion. Toward the end of the pseudopregnancy (psp) resulting from a sterile mating or artificial stimulation of the uterine cervix, ovarian estradiol secretion is enhanced, and luteal progesterone (P) secretion wanes. This shifting ratio terminates the surges of PRL by day 13 of psp. Ovariectomy on day 8 of psp and simulation of this pattern of steroid secretion with sc Silastic implants also resulted in termination of surges by day 13. However, ovariectomy on days 2, 4, or 6, followed immediately by implantation of the inhibitory regimen of ovarian steroids, did not result in termination of the noctural surge by days 7, 9, or 11, respectively. The diurnal surge was absent in all cases. A third and fourth CS applied on days 7 and 8 of psp were not able to overcome the effects of the inhibitory steroid regimen begun on day 8. PRL surges induced in ovariectomized rats by one 30-sec CS persist for 6 days (suboptimal stimulation), while surges induced by two 30-sec CS persist for at least 10 days (optimal). However, implantation o P or hysterectomy permits suboptimally stimulated ovariectomized rats to secrete nocturnal surges through day 10. These data indicate that optimal CS programs the PRL-releasing apparatus to secrete surges for a preset interval, during which time the system remains unresponsive to physiological inhibitory and excitatory input. However, suboptimal CS can be reinforced by imposition of P or removal of PRL inhibitory activity of uterine origin.

An ovarian role in prolonging and terminating the two surges of prolactin in pseudopregnant rats.

Daily nocturnal (N) and diurnal (D) surges of PRL are characteristic of a pseudopregnancy (psp) induced by cervical stimulation (CS). These semicircadian surges persist for 13-14 days, after which time they cease, and psp ends. The purpose of this study was to describe the involvement of the ovarian steroids in the maintenance and termination of the N and D surges of PRL. CS of long term ovariectomized (OVX) rats resulted in N and D surges of PRL lasting 14 days. By day 16 after CS, neither surge of PRL was present. The sc placement of Silastic implants containing large quantities of progesterone in long term OVX-CS rats sustained and enhanced the N surge of PRL until at least day 16. The D surge was also sustained until day 16, but its magnitude was normal. Estradiol alone was capable of maintaining the D but not the N surge for this same time interval. However, pharmacological quantities of estradiol restricted the magnitude of the N surge generated in the progesterone-implanted animal. Since neither surge of PRL was present by day 14 after CS in intact rats, but at least the nocturnal surge was still present at this time in long term or acutely OVX rats, this implies an active role of ovarian steroids in the termination of PRL surges at the end of psp. At the end of psp, luteal progesterone secretion declines and follicular estradiol secretion increases. Varying sizes of Silastic implants containing progesterone or estrogen were placed into psp animals after acute ovariectomy on day 9 to approximate this ovarian steroid secretory pattern of intact psp rats. Only when the decrease in progesterone was coupled with a modest increase in estrogen was there a total cessation of the two daily surges of PRL in OVX-CS rats. The data suggest that the PRL surges end in the intact psp rat due to the waning response to CS as well as the shifting steroid ratio represented by the fall in progesterone in the presence of a small amount of estrogen.

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.

Endothelin activates large-conductance K+ channels in rat lactotrophs: reversal by long-term exposure to dopamine agonist.

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nM ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nM ET-1 on these channels was markedly attenuated in the presence of the selective ET(A) receptor antagonist BQ-610 (200 nM), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mM KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 microM Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BK(Ca)) channels were blocked by 20 mM tetraethylammonium but were insensitive to the K+ channel blockers apamin (1 microM), charybdotoxin (200 nM), or iberiotoxin (200 nM). Acute application of 10 microM dopamine and 20 nM ET-1 caused activation of BK(Ca) channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (+/-)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nM), bath application of 20 nM ET-1 resulted in inhibition of spontaneously active BK(Ca) channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BK(Ca) channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ET(A) receptor to BK(Ca) channels.

Endothelin is an autocrine regulator of prolactin secretion.

The aim of this study was to establish the cellular source of ET-like peptides affecting PRL secretion. Fluorescence double label immunocytochemistry and confocal laser scanning microscopy were used to demonstrate cellular colocalization for PRL and endothelin-1 (ET1)-like immunoreactivities in the anterior lobe of the pituitary gland of rats. An ET-specific reverse hemolytic plaque assay was applied to demonstrate that lactotrophs are capable of releasing ET-like peptides. A PRL-specific reverse hemolytic plaque assay was used to assess the influence of the released endogenous ETs on PRL secretion. ET(A)-specific receptor antagonists BQ123 and BQ610, and endothelin convertase enzyme inhibitory peptide, [22Val]big ET1-(16-38), increased PRL secretion, whereas the ET(B) receptor-specific antagonist BQ788 was ineffective. The ET(A) antagonist BQ123-induced increase in PRL secretion followed a bell-shaped dose-response curve in cells obtained from female rats, whereas it followed a sigmoid curve in males. Frequency distribution of PRL plaque sizes using logarithmically binned data revealed two subpopulations of lactotrophs with differential responsiveness to endogenous ETs. These data demonstrate that a large proportion of lactotrophs is capable of expressing and secreting ET-like peptides in biologically significant quantities. As low pituitary cell density in reverse hemolytic plaque assay minimizes cell to cell communications, these findings constitute direct proof of autocrine regulation of PRL secretion by ET-like peptides.

Long term exposure to dopamine reverses the inhibitory effect of endothelin-1 on prolactin secretion.

This study was undertaken to assess the possibility that endothelin-1 (ET-1) and dopamine (DA) can act in concert to modulate PRL secretion. Enzymatically dispersed anterior pituitary cells obtained from random cycling female rats were perifused with Dulbecco's Modified Eagle's Medium supplemented with 0.2% BSA and 100 microM ascorbic acid. In the absence of dopamine, ET-1 (applied at 20 nM for 60 min) rapidly evoked a small transient elevation of PRL release, followed by a sustained inhibitory phase. Overnight perfusion with 500 nM DA-supplemented medium did not change the basic character of ET-1's effects on PRL secretion. Continuation of DA exposure for 48 h dramatically shifted the responsiveness of the lactotrophs to ET-1; the fast stimulatory response was robustly enhanced, whereas the inhibitory phase was replaced by a modest secondary elevation of basal PRL secretion. The stimulatory effect of ET-1 on PRL secretion after DA pretreatment was blocked by an ETA receptor antagonist, BQ-123. The effect of DA can be mimicked completely by a specific D2 receptor agonist (+/-)-2-(N-phenyl-N-propyl)amino-5-hydroxytetraline hydrochloride, whereas pretreatment with a D1 agonist, SKF-39393, failed to change the responsiveness of lactotrophs to ET-1. Our data indicate that persistent activation of D2 receptors, a condition most closely resembling the in vivo environment of the lactotrophs, uncouples the inhibitory signaling pathway from the ETA receptor while synergistically affecting signal transduction, which mediates the ET-induced stimulation of PRL secretion.