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M E Freeman

Publications and source records attributed to M E Freeman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ovarian steroids differentially regulate the expression of PRL-R in neuroendocrine dopaminergic neuron populations: a double label confocal microscopic study.

The aims of this study were (1) to identify the possible hypothalamic targets for a short prolactin (PRL) feedback in the adult female rat by identifying DAergic neuron populations expressing PRL receptor (PRL-R); (2) to describe the effect of ovarian steroids on the expression of PRL-R and (3) to compare the distribution of both the extracellular (EC) and ligand binding (LB) domains of the PRL-R on the hypothalamic dopaminergic neurons by applying double label immunocytochemistry for the different domains of PRL-R and for tyrosine hydroxylase (TH). Five- to six-month-old female rats were ovariectomized (OVX) and implanted with either 17 beta-estradiol (E2), progesterone (P4) or received an E2 and a P4 implant (E2 + P4) at the same time. In the periventricular nucleus and in the dorsomedial portion of the middle arcuate nucleus, a dramatic increase in PRL-REC immunoreactivity was observed in E2 implanted rats. This increase was attenuated in E2 + P4 rats, but P4 treatment alone had no effect. Changes in PRL-REC expression were paralleled by changes in serum PRL levels. Interestingly, PRL-REC expression in the rostral arcuate nucleus decreased in P4 implanted rats, however, P4 did not attenuate the E2-induced increase in PRL-REC density. PRL-REC immunostaining was observed on the membrane, in the cytoplasm and in the nucleus. PRL-RLB immunoreactivity was also detectable in the TH positive neurons, but no nuclear staining was observed with this antibody. However, we found a strong PRL-RLB immunostaining in the ependymal lining of the 3rd ventricle and in the processes of tanycytes projecting to the median eminence. These data indicate that (1) all neuroendocrine DAergic cells can be targets for PRL, (2) expression of PRL-R is differentially affected by ovarian steroids in the different TH cell populations, (3) PRL-RLB domain may be involved in trafficking PRL in the median eminence.

Animals↗
PubMed1998

Changes in the local metabolism of dopamine in the anterior and neural lobes but not in the intermediate lobe of the pituitary gland during nursing.

Dopamine (DA), produced by tubero-infundibular dopaminergic (TIDA) neurons of the arcuate nucleus (ARN) is the established inhibitor of the secretion of prolactin (PRL). Changes in dopaminergic (DAergic) neuronal activity in the median eminence-long portal vessels (ME-LPV) and/or the concentration of DA in the anterior lobe (AL) are inversely related to the secretion of PRL. However, conflicting reports concerning DAergic neuronal activity during the suckling-induced release of PRL persist. In addition to TIDA neurons, PeVN-hypophysial DAergic (PHDA) and tubero-hypophysial DAergic (THDA) neurons which, respectively, innervate the intermediate lobe (IL) and the IL/neural lobe (NL) also have a significant role. We measured the concentrations of DA and its main metabolite, 3, 4-dihydroxyphenylacetic acid (DOPAC), in the median eminence and the three pituitary lobes of lactating mothers. Concentrations of DA and DOPAC from tissues and the concentration of PRL in plasma were measured by HPLC-EC and RIA, respectively. There were no changes in the concentration of DA and DOPAC of the IL due to the suckling stimulus. In the NL, a decrease in the concentration of DOPAC was detected due to the suckling stimulus. In addition, there were no changes of DA or DOPAC in the outer zone of the AL (AL-OZ) due to suckling. However, a decrease in the concentrations of DA and DOPAC was detected in the inner zone of the AL (AL-IZ). These data suggest lactotrophs from the AL-IZ are responsible for the changes in the concentration of plasma PRL in response to the suckling stimulus. In addition TIDA and THDA neurons, but not PHDA neurons, regulate the control of the secretion of PRL in response to suckling.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1998

Characterization of the dopaminergic input to the pituitary gland throughout the estrous cycle of the rat.

Changes in the concentrations of dopamine (DA) and its major metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were characterized in the pituitary gland throughout the 4-day estrous cycle of the rat. Female rats were sacrificed at 2- to 3-hour intervals throughout each day of the 4-day estrous cycle. Pituitary glands were removed, and the concentrations of DA and DOPAC were determined by high-performance liquid chromatography coupled to electrochemical detection. The concentration of prolactin (PRL) in serum from these same animals was determined by radioimmunoassay. The concentration of DA in the anterior lobe was constant throughout most of the 4-day estrous cycle. Prior to initiation of the proestrous surge of PRL, there were significant (p < 0.05) decreases in the concentrations of both DA and DOPAC in the anterior lobe which returned to elevated baseline levels just prior to the termination of the proestrous surge of PRL. The concentrations of DA and DOPAC in the intermediate lobe exhibited a daily rhythm. However, in the intermediate as well as in the anterior lobe, there were significant (p < 0.001) decreases in the concentrations of both DA and DOPAC, coincident with the initiation of the proestrous surge of PRL. Similarly, coincident with the peak of the proestrous surge of PRL, there were significant (p < 0.001) increases in the concentrations of DA and DOPAC in the intermediate lobe, followed by a return to basal levels and resumption of the daily rhythm. The pattern of the concentrations of DA and DOPAC in the neural lobe was also daily in nature, with peaks occurring between 13.00 and 15.00 h each day of the 4-day estrous cycle. These data, taken together: (1) confirm that a decrease of the concentrations of DA and DOPAC occurs in the anterior lobe prior to the proestrous surge of PRL; (2) reveal that DA is released in a daily pattern at intermediate and neural lobes, and (3) suggest an apparent role for DA released to the intermediate lobe in the regulation of the proestrous surge of PRL.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1998

Ovarian steroids modulate responsiveness to dopamine and expression of G-proteins in lactotropes.

The effects of chronic ovarian steroid treatment on the secretory activity of individual lactotropes and the mechanisms modulating their responsiveness to dopamine (DA) were studied. Female rats were ovariectomized (OVX) and implanted with Silastic capsules containing progesterone (P4), 17beta), 17beta-estradiol (E2) or both E2 (E2+P4). Ten days after surgery, anterior pituitaries were enzymatically dispersed and the reverse hemolytic plaque assay (RHPA) was performed to assess the release of prolactin (PRL) from individual lactotropes. RHPA was combined with immunocytochemistry (ICC) for PRL, Galphas or Gialpha3/Galphao proteins. E2 treatment alone or in combination with P4 increased the percentage of immunoreactive lactotropes among anterior pituitary cells. Incidence of active (plaque-forming) lactotropes, however, was increased both in P4-, and E2-treated rats and E2+P4 treatment increased it even further. While P4 treatment did not affect the frequency distribution of lactotropes, both E2 and E2+P4 treatments increased the large plaque-forming lactotrope population. This increase was reflected by the significantly greater mean plaque areas of lactotropes from E2- and E2+P4-treated rats compared to OVX or P4-treated animals. The responsiveness of lactotropes to DA from P4-treated rats did not differ from that of OVX rats: thus challenge with 1 microM DA inhibited the release of PRL, while 100 pM DA had no effect. E2 and E2+P4 treatments, however, profoundly changed the lactotrope's responsiveness: challenge with 1 microM DA had no effect and 100 pM DA resulted in moderate stimulation of PRL release in E2+P4 rats. Double-label ICC revealed that ovarian steroid treatments did not affect the expression of Galphas in lactotropes. The incidence of Gialpha3/Galphao-immunoreactive lactotropes, however, decreased after E2 treatment, alone or in combination with P4. Although expression of Galphas was similar in all plaque-forming cells regardless of plaque size, lactotropes expressing Gialpha3/Galphao were more likely to form small plaques in all treatment groups. These data suggest: (1) ovarian steroid treatment recruits quiescent lactotropes to release PRL; (2) E2 treatment alone or in combination with P4 increases the amount of PRL rleased by individual lactotropes; (3) E2-induced alterations in the frequency distribution and lactotrope responsiveness to DA may be due in part to a decreased expression of Gialpha3/Galphao.

Animals↗
PubMed1998

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.

Animals↗
PubMed1997

Effects of ovarian steroid hormones on dopamine-controlled prolactin secretory responses in vitro.

Dopamine (DA), a well-established inhibitor of prolactin (PRL) secretion, has also been shown to stimulate PRL secretion from the anterior lobe of the pituitary gland of the rat. It has been reported that low doses of DA stimulate PRL whereas high doses inhibit PRL secretion. Our laboratory has previously reported that these PRL secretory responses are dependent upon the stages of the estrous cycle of the rat. The objective of the present study was to determine the steroid requirements for the differing PRL secretory responses to low and high doses of DA in perifusion. Animals were ovariectomized (OVX) and immediately given Silastic implants containing estradiol (E2) which has previously been shown in our laboratory to produce blood levels of 70-100 pg/ml, progesterone which has previously been shown in our laboratory to produce blood levels of 30-40 ng/ml, or the combination. OVX rats served as controls. Ten days later, the anterior lobes of the pituitary glands were harvested and enzymatically dissociated. Cells were mixed with Sephadex G-10 and placed in six 0.5-ml perifusion chambers (1 x 10(6) cells/chamber). Cells were perifused for 24 h with Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 0.1 mM ascorbic acid. The PRL secretory pattern was characterized in response to the following treatment sequence: (1) 30 min media alone (maximally uninhibited); (2) 24 min 100 pM DA; (3) 30 min media alone (DA withdrawal); (4) 24 min 1 microM DA, and (5) 30 min media alone (DA withdrawal). PRL secretion in the presence of 100 pM DA was unchanged in cells obtained from OVX animals, but exposure to 1 microM DA inhibited PRL release in these cells. Subsequent withdrawal stimulated PRL secretion relative to that of the initial exposure to media alone. The responses of cells from OVX rats implanted with progesterone alone was indistinguishable from those of the OVX controls. In cells obtained from animals implanted with E2 alone, 100 pM DA and its subsequent withdrawal neither stimulated nor inhibited PRL secretion. In contrast, 1 microM DA exposure initially stimulated and then inhibited PRL secretion in cells from E2-treated animals. Here, subsequent withdrawal of DA enhanced PRL secretion. In cells obtained from E2+progesterone-treated animals, 100 pM DA exposure robustly enhanced PRL secretory responses. Withdrawal of this dose of DA had no further effect on PRL secretion. However, exposure of E2+progesterone-treated cells to 1 microM DA robustly stimulated and subsequently only slightly inhibited PRL secretion. The results of these studies suggest that inhibition of PRL secretion by DA is independent of ovarian steroids while E2 and progesterone in combination favors stimulation of PRL secretion in response to DA. Taken together, these data suggest that PRL secretory responses in this system are determined by the ovarian steroid milieu.

Animals↗
PubMed1997

Dopaminergic neurons in periventricular and arcuate nuclei of proestrous and ovariectomized rats: endogenous diurnal rhythm of Fos-related antigens expression.

The expression of Fos-related antigens (FRAs) in the A12 and the A14 hypothalamic dopaminergic neurons was compared throughout the day in proestrous (PRO) and ovariectomized (OVX) rats to establish the relationship between secretion of prolactin (PRL) and dopaminergic neuronal activation. Animals with intact ovaries were sacrificed at 11:00, 13:00, 15:00, 17:00 and 21:00 h on the day of proestrus and 06:00 and 09:00 h in the morning of estrus. OVX animals were sacrificed at the same time points on the 12th day after surgery. Double-label immunocytochemistry was performed by using antibodies against FRAs as markers of tonic neuronal activity and tyrosine hydroxylase to identify dopaminergic neurons. Serum PRL levels were determined by radioimmunoassay. A pattern of FRAs expression was present in the A14 and A12 neurons of PRO rats. The incidence of FRAs expressing neurons was the highest in the first half of the day when the PRL levels were low, and decreased prior to the surge of serum PRL during the afternoon. This pattern was present in the dopaminergic neurons of the periventricular nucleus (A14), and in all portions of the arcuate nucleus (A12) except the ventrolateral portion of the middle arcuate nucleus. A similar pattern of FRAs expression existed in the A14 and A12 neurons of OVX rats in spite of that there were no detectable changes in serum PRL levels. However, the amplitude of decrease in the incidence of FRAs-labeled neurons was lower in OVX than in PRO rats. These data suggested that similar to the control of hypothalamic PRL releasing factors, the activation/deactivation pattern of the A12 and the A14 neurons is governed by a seemingly endogenous rhythmic input. The incidence of this rhythm is independent of the reproductive state, but its amplitude is enhanced by ovarian steroids. The disparity between the FRAs expression and serum PRL levels in OVX rats indicated that the effect of this endogenous dopaminergic rhythm upon PRL secretion is dependent on the ovarian steroid background.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

Dopaminergic neurons of periventricular and arcuate nuclei of pseudopregnant rats: semicircadian rhythm in Fos-related antigens immunoreactivities and in dopamine concentration.

The activity of the A14 and A12 hypothalamic dopaminergic (DAergic) neurons were characterized throughout a day of pseudopregnancy to establish their relationship to the generation of PRL surges during that time. Double-label immunocytochemistry was performed using fos-related antigens as markers of neuronal activity, and tyrosine hydroxylase antibody to identify DAergic neurons of the periventricular nucleus and the arcuate nucleus (ARN). Dopamine concentrations were measured with HPLC with electrochemical detection from micropunched samples to approximate synthetic activities of A14 and A12 neurons. Serum PRL levels were determined by RIA. On the fifth and sixth day of pseudopregnancy, five rats/time point were killed at 1100 h, 1300 h, 1500 h, 1800 h, 2100 h, 2400 h, 0300 h, and 0600 h. When the serum PRL was low, the incidence of fos-related antigens/tyrosine hydroxylase double-labeled neurons was high, and it decreased twice a day by 1500 and 0300 h in the periventricular nucleus, rostral ARN, dorsomedial subpopulation of the middle ARN, and in the caudal ARN, corresponding to the initiation of PRL surges. Concentrations of DA were high in these areas, and decreased before PRL surges in agreement with the immunocytochemical data. These findings suggest a semicircadian rhythm in the activities of A14 and A12 neurons which, in concert with PRL releasing factors, contribute to the generation of PRL surges in pseudopregnant animals.

Animals↗
PubMed1995

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.

Alleles↗
PubMed1995

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.

Animals↗
PubMed1994

A transformed murine Leydig cell line expresses the ETA receptor subtype.

We recently demonstrated that transformed murine Leydig cells (MA-10) responded to endothelin-1 (ET-1) via increased steroidogenesis. This study addresses the endothelin receptor subtype present on this cell line and whether or not the cells produce ET-1. The expression of the preproendothelin-1 (PPET-1) gene was investigated by Northern blot analysis, and PPET-1 mRNA was found to be < 0.2% of that present in pulmonary endothelial cells. The medium from MA-10 cells, maintained under serum-free conditions, was analyzed by radio-immunoassay to determine immunoreactive-ET-1 production and ET-1 levels were found to be below the sensitivity of the assay (< 10 pg/ml). The data from competitive binding experiments with [125I]ET-1 and unlabeled ET-1, ET-3 and receptor subtype selective ligands yielded a single class of high affinity binding sites with ETA receptor subtype characteristics. The results of this study demonstrate that MA-10 cells possess the ETA receptor subtype but do not produce significant quantities of ET-1 under basal conditions.

Animals↗
PubMed1994

Comparison of the forms of the dopamine D2 receptor expressed in GH4C1 cells.

The effects of dopamine (DA) on prolactin (PRL) secretion, phosphoinositide metabolism, cytosolic calcium concentrations ([Ca2+]i), and cAMP production in GH4C1 cells expressed either the short (GH4ZR7 cells) or long (GH4I12 cells) form of the rat DA D2 receptor were compared in this study. The GH4C1 cell line is derived from the rat anterior pituitary gland and lacks functional DA receptors. The GH4ZR7 and GH4I12 cell lines have been transfected with either the short or the long form of the D2 receptor, respectively. In this study, the functional coupling of these receptors to both basal and stimulated PRL secretion and to common second messenger systems was examined. Both cell types expressing DA receptors exhibited similar saturable binding to the D2 antagonist [3H]spiperone (Kd GH4ZR7 = 96 +/- 8 pM, Kd GH4I12 = 107 +/- 49 pM). In GH4ZR7 cells, 1 and 10 microM DA inhibited basal PRL secretion by 37% and 58%, respectively. In GH4I12 cells, 1 and 10 microM DA inhibited basal PRL secretion by 63% and 54%, respectively. In GH4ZR7 cells, 10 microM DA completely reversed the stimulatory effects of 1-100 nM thyrotropin-releasing hormone (TRH), and 1 microM DA attenuated the stimulatory effects of 10 and 100 nM TRH. Interestingly, GH4I12 cells were not responsive to TRH. In both cell lines, the inhibitory effects of DA were blocked by the specific D2 antagonist, eticlopride. The stimulatory effects of TRH on [Ca2+]i were dose dependent and could be blocked (at least in GH4ZR7 cells) by prior treatment of the cells with 1 microM DA. The ability of dopamine to block the TRH-mediated increase in [Ca2+]i was attenuated by eticlopride. DA (1 microM) had no effect on resting [Ca2+]i in either cell line expressing DA receptor. TRH (100 nM) maximally stimulated total inositol phosphate (IP) accumulation to values approximately three times greater than controls in GH4C1 and GH4ZR7 cells only. DA had no effect on basal or TRH-stimulated IP accumulation in any of the cell lines. DA (1 and 10 microM) inhibited cAMP production by 40% and 39%, respectively, in GH4ZR7 cells. Similarly, in GH4I12 cells, DA (1 and 10 microM) inhibited cAMP production by 48% and 60%, respectively. These data indicate that both the long and short forms of the D2 receptors play similar roles when expressed in GH4C1 cells. However, maximal inhibition of PRL in these cell lines is approximately 20% less than that of normal lactotrophs.

Animals↗
PubMed1993

Characterization of endothelin receptors in the anterior pituitary gland.

To characterize endothelin (ET) receptors modulating pituitary hormone secretion, potencies of ET-like agonists were compared on prolactin (PRL), thyrotropin (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) secretion from primary cultures of female rat pituitary cells. ET-1 was more potent than ET-3 in all cases. Sarafotoxin (SRTX) S6b an ETA agonist, was also more potent than ET-3 in all cases. SRTX-c, an ETB receptor agonist, was inactive. The ET-1-to-ET-3 potency ratio was three orders of magnitude higher on PRL or TSH secretion than on LH and FSH secretion, whereas SRTX-b-to-ET-3 potency ratios were similar on all four hormones. The ETA antagonist BQ-123 caused a parallel dextral displacement of dose-response curves of ET-1 and ET-3 on all four hormones. Schild regressions for BQ-123 on ET-1-induced PRL, TSH, LH, and FSH secretion indicated that BQ-123 has a similar affinity for the receptors mediating ET-1's effects. When BQ-123 was assessed against ET-3, Schild regressions indicated greater affinity for ET-3 on lactotrophs and thyrotrophs than gonadotrophs. Thus changes in pituitary hormone secretion are mediated by ETA-like receptors. ET receptors in lactotrophs and thyrotrophs are clearly distinguishable from gonadotrophs. We suggest the existence of distinct ETA receptor subtypes (ETA1 and ETA2) on these differing pituitary cells.

Animals↗
PubMed1993

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+.

Animals↗