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L S Frawley

Publications and source records attributed to L S Frawley.

At least 91 records · Page 5Linked to original sources
PubMed1987

Evidence for the existence of a luteal cell type that is steroidogenic and releases relaxin.

Secretion of relaxin from cultured luteal cells derived from pregnant sows was detected by a reverse hemolytic plaque assay. In this method, luteal cells are cultured in monolayers together with protein-A-conjugated ovine red blood cells. In the presence of porcine relaxin anti-serum and complement, relaxin-releasing cells become surrounded by an area of hemolysis--a plaque--which can be microscopically visualized. After fixation, these same luteal cells in monolayers were stained for the presence of 3 beta-hydroxysteroid dehydrogenase, an enzyme marker for steroidogenic cells. Cells could then be classified by their ability to form plaques (relaxin-releasing cells) and/or steroidogenic capability (positive staining). Dual-secretors (large luteal cells that were steroidogenic and released relaxin) could be identified in dispersed luteal cells derived from pigs at all stages of pregnancy examined (Day 22-112 of gestation, n = 9; term is Day 114 +/- 2 days). In addition, luteal cells were detected that were either steroidogenic only or released relaxin, and finally, cells that appeared to possess neither endocrine capability. Frequency analysis of functional subtypes indicated approximately equal representation of each in the first half of pregnancy, but an apparent fall in relaxin-releasing cells in the preparturient period. It is suggested that dual-secretors may represent one mechanism that allows the corpus luteum to express multiple endocrine function during pregnancy without the requirement for increased cell numbers.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Estradiol induces a shift in cultured cells that release prolactin or growth hormone.

The process by which estradiol induces an increase in the percentage of prolactin cells in the rat pituitary requires clarification. In this study the effect of estradiol treatment on growth hormone and prolactin cells in monolayer cultures of male rat pituitaries was assessed by use of reverse hemolytic plaque assays. Treatment of cultures with 17 beta-estradiol (10 nM) for 6 days elevated significantly (P less than 0.01) the percentage of all cells in culture that released prolactin without affecting the percentage of growth hormone secretors. However, by sequentially assaying single cells for growth hormone and prolactin, we found that estradiol caused a marked increase (P less than 0.01) in the proportion of individual cells that released both growth hormone and prolactin and a commensurate decrease in the proportion that released growth hormone alone. These results demonstrate that estradiol acts directly on cultures of male pituitary cells to shift the proportions of cells that released growth hormone, prolactin, or both hormones and indicate that estradiol may convert cells that release only growth hormone to those that release both growth hormone and prolactin.

Animals↗
PubMed1986

Analysis by plaque assays of GH and prolactin release from individual cells in cultures of male pituitaries. Evidence for functional heterogeneity within rat mammotrope and somatotrope populations.

In this study we evaluated the quantitative influence of GRF and TRH on the rate of hormone secretion from single cells in cultures of male pituitaries. To accomplish this, we dispersed pituitaries from male rats with trypsin and cultured them for 24 or 48 h. Reverse hemolytic plaque assays for GH and prolactin were then performed on retrypsinized cultures to identify individual cells that secreted these hormones. Mammotropes and somatotropes were found to comprise 31.4 +/- 1.8 and 32.2 +/- 0.9% (mean +/- SE, n = 3 experiments), respectively, of all cells in 24-hour cultures. Immunocytochemical staining of different batches of cells from the same dispersions corroborated the proportions of these two cell types. Differences in the rate of basal hormone secretion were observed within each of these cell populations as evidenced by the gradual appearance of prolactin and GH plaques over a 4-hour period when incubations were conducted in the absence of stimulatory secretagogues. Addition of increasing concentrations of GRF (1 X 10(-10) -1 X 10(-7) M) or TRH (1 X 10(-9) -1 X 10(-6) M) to these incubations resulted in dose-related increases in the rate of GH and prolactin plaque formation, respectively. Maximal plaque development by somatotropes could be induced within 30 min of administering large doses of GRF, indicating that most, if not all somatotropes are responsive to this secretagogue. In contrast, approximately one third of all mammotropes could not be stimulated to form plaques acutely when subjected to similar treatment with TRH. This observation suggests that mammotropes are heterogeneous with respect to TRH responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Capacity of individual somatotropes to release growth hormone varies according to sex: analysis by reverse hemolytic plaque assay.

A reverse hemolytic plaque assay was used to investigate the mechanism(s) underlying sexual differences in GH release which evolve at puberty in rats. The percentages of GH-secreting cells in 24-h pituitary cultures from each sex were similar for pituitary donors up to 30 days of age (range = 38.9% to 41.7% of all cells in culture, n = 3 separate experiments) but decreased by day 50. The decrease was more striking for females (to 24.1 +/- 0.3% mean +/- SE) than for males (to 33.2 +/- 1.1%). However, owing to the greater increase in total pituitary cell number exhibited by female rats at this time, the absolute numbers of somatotropes recovered from male and female pituitaries were almost identical on 50 and 100 days of age. To assess the secretory capacities of individual somatotropes, we measured the sizes of plaques formed. In prepubertal rats (days 10-30), the plaque areas under basal conditions were comparable for males and females at each age studied, and treatment with GH-releasing factor increased plaque sizes to approximately the same degree (10-fold) for both sexes at each age. However, by day 100, plaques that formed under both basal and stimulated conditions were consistently larger (P less than 0.01) for male than for female donors. Taken together, our results demonstrate that sexual differences in GH release are attributable to the secretory capacities of individual somatotropes rather than to differences in the numbers of GH cells in pituitaries of male and female rats.

Animals↗
PubMed1986

Ovine prolactin (PRL) and dopamine preferentially inhibit PRL release from the same subpopulation of rat mammotropes.

Autoregulation of PRL release was studied at the single cell level by the use of a reverse hemolytic plaque assay. Monodispersed pituitary cells from adult male rats were first preincubated with test substances and then coincubated with antirat PRL antiserum before development of plaques with complement. At the conclusion of the assay, the percentage of all pituitary cells in culture that formed plaques was evaluated microscopically, and the rate of plaque development was used as an index for the rate of hormone release. In controls, the maximal percentage of pituitary cells formed PRL plaques within a 1.5-h antibody incubation period, and addition of TRH (1 X 10(-7) M) did not increase this proportion. Treatment with ovine PRL (oPRL, 100 ng/ml) or dopamine (1 X 10(-7) M), either alone or in combination, caused a comparable suppression of the rate of PRL plaque development, which was reversed by the presence of TRH. Pretreatment of cells with the lysosomotropic agent chloroquine (1 X 10(-5) M) overrode dopamine inhibition of PRL plaque development, but did not influence oPRL inhibition. Taken together, these results demonstrate that oPRL inhibits basal, but not TRH-induced, PRL release from rat pituitary cells and support the view that PRL can act at the pituitary level to inhibit its own secretion. Moreover, the equipotency and lack of additivity exhibited by oPRL and dopamine coupled with the differential effects of chloroquine suggest that these factors both act upon the same subpopulation of mammotropes to inhibit PRL release, but by separate intracellular mechanisms.

Animals↗
PubMed1986

A novel bioassay for lactogenic activity: demonstration that prolactin cells differ from one another in bio- and immuno-potencies of secreted hormone.

A reverse hemolytic plaque assay for detecting casein release from individual mammary cells in culture was developed as a bioassay for PRL. Treatment with rat PRL caused dose-related increases in the percentage of mammary cells that released casein and the average size of casein plaques that formed. The assay exhibited exquisite sensitivity (156 fg rat PRL per assay slide) and could be used to evaluate the biopotency of PRL released from individual cells. By combining this "plaque bioassay" with a standard plaque assay for measuring the secretion of immunoreactive PRL, it was possible to compare the bio- and immuno-potencies of hormone released from the same pituitary cells. The results of three separate studies revealed major differences among PRL secretors in these potency estimates. Given the existence of PRL variants with different biological and immunological efficacies, these findings suggest that PRL cells differ from one another in the molecular form(s) of hormone released.

Animals↗
PubMed1985

Oxytocin attenuates TRH-induced TSH release from rat pituitary cells.

The observation that suckling evokes a modest rise in serum TSH when compared with that of prolactin is inconsistent with the hypothesis that TRH serves as a hypophysiotropic mediator of this response. In the present study we attempted to provide an explanation for this discrepancy by determining whether any of a growing number of putative prolactin releasing factors could alter pituitary responsiveness to TRH. Anterior pituitaries from lactating (day 14) rats were monodispersed with trypsin, cultured for 2 days, and then incubated in the presence of medium alone or medium containing TRH, dopamine, or a combination of these secretagogues. Companion sets of cultures were incubated concurrently with either beta-endorphin, neurotensin, oxytocin, serotonin, vasoactive intestinal polypeptide, or lysine vasopressin. As expected, TRH stimulated and dopamine suppressed prolactin release. None of the substances tested except oxytocin had a significant effect on pituitary cell responsiveness to TRH or dopamine. Oxytocin had no effect on prolactin secretion when tested alone or in combination with TRH and dopamine. TRH alone stimulated TSH release by these cultures, while oxytocin and dopamine were ineffective by themselves. However, TSH secretion by cultures treated simultaneously with TRH and oxytocin could be suppressed to approximately half of that released by cells incubated with TRH alone. These results demonstrate that oxytocin attenuates TRH-induced TSH release by a direct action on pituitary cells without affecting the prolactin response. This selectivity of responsiveness imparted by oxytocin might contribute to the blunted release of TSH after suckling.

Animals↗
PubMed1985

Identification by plaque assays of a pituitary cell type that secretes both growth hormone and prolactin.

Sequential application of reverse hemolytic plaque assays for GH and PRL revealed the presence of individual pituitary cells that released both hormones. These dual cells accounted for approximately one third of all GH and/or PRL secretors in 24-h pituitary cultures derived from male rats. Additional studies in which a different version of the plaque assay and double-staining immunocytochemistry were applied separately to dispersed pituitary cells from males yielded results that were virtually identical. These results suggest that mammosomatotropes, cells that secrete both GH and PRL, may exist in pituitaries of normal rats.

Animals↗
PubMed1985

Functional maturation of somatotropes in fetal rat pituitaries: analysis by reverse hemolytic plaque assay.

Immunocytochemistry (ICC) and a reverse hemolytic plaque assay for GH were used to investigate the temporal relationships between the initiation of hormone storage and release by developing somatotropes and the onset of responsiveness of these cells to stimulatory and inhibitory secretagogues. Anterior pituitaries obtained from rats on days 18-21 of fetal development (pups were generally delivered on fetal day 22, which is equivalent to day 0 of neonatal life) were monodispersed with trypsin, cultured for 24 h, and then subjected to reverse hemolytic plaque assay and/or ICC for GH. GH-containing cells (determined by ICC) were extremely rare (less than 1%) in cultures derived from day 18 fetuses, but accounted for 22.4%, 25.2%, and 24.5% of all cells in cultures from day 19-21 fetuses, respectively. The proportion of GH-releasing cells, as determined in a long term (120-min incubation with antibody) plaque assay, was less than 1%, 22.4%, and 22.9% for days 18, 20, and 21, respectively, but only 13.6% for day 19 cells. Thus, many pituitary cells from day 19 fetuses contained, but did not release, GH. While GH-releasing factor (1-44) (1 X 10(-7) M) had no effect on the percentage of GH plaque-forming cells in long term incubations, it enhanced (by approximately the same degree in day 19-21 groups) the percentage of cells that formed plaques and the size of the plaques in short term (45-min) incubations with antibody. Somatostatin (1 X 10(-7) M) exerted inhibitory effects on these variables when tested in long term incubations, and age of the donor rats did not influence pituitary responsiveness to this secretagogue. These results suggest that the capacities of fetal somatotropes to store GH and release it under basal and regulated conditions are attained, in large part, within an extremely narrow time frame between days 18 and 19 of fetal development.

Animals↗
PubMed1985

Ontogeny of prolactin cells in neonatal rats: initial prolactin secretors also release growth hormone.

Reverse hemolytic plaque assays and immunocytochemistry were used to monitor the ontogeny of individual hormone-secreting and hormone-containing cells in rats. Monodispersed anterior pituitary cells from fetal rats (sex unspecified) and neonatal rats of each sex were cultured for 24 h and then subjected to immunocytochemistry or plaque assays for PRL or GH. PRL secretors first appeared in appreciable numbers in cultures from 4-day-old animals, and by day 5, they accounted for 8-12% of all cells in culture. The percentage of GH secretors rose to a peak on day 5 (comprising approximately 40% of all cells), when the values were slightly higher than those observed previously in adults. The percentage of cultured cells from 4- to 5-day-olds that released PRL or GH was not influenced by the sex of the donor animal and was consistent with immunocytochemical estimates. Using a sequential plaque assay that enabled the detection of both GH and PRL release from the same cells, we found that of every 100 pituitary cells from 5-day-old males that released PRL and/or GH 62.5 released GH only, 1.7 released PRL alone, and the remaining 35.8 released both hormones. Almost identical proportions were found for females. These findings were confirmed using an additional variation of the plaque assay and by double staining immunocytochemistry. Taken together, these results indicate that mammosomatotropes, cells that release both GH and PRL, appear early in the neonatal development of both sexes and raise the possibility that PRL-secreting cells arise from GH-secreting cells.

Animals↗
PubMed1985

Cultures of GH3 cells are functionally heterogeneous: thyrotropin-releasing hormone, estradiol and cortisol cause reciprocal shifts in the proportions of growth hormone and prolactin secretors.

Utilization of reverse hemolytic plaque assays revealed that cultures of GH3 cells are not functionally homogeneous, but contain approximately twice as many GH as PRL secretors. Chronic treatment of these cultures with TRH, estradiol, or cortisol (factors that induce reciprocal alterations in the amount of GH and PRL released by entire cultures of GH3 cells) caused reciprocal shifts in the proportions of GH and PRL cells, without influencing the combined percentages of hormone-secreting cell types present. These results indicate that alterations in hormone release caused by these modulatory factors may in part be due to changes in the ratios of cells committed to the secretion of each hormone. Moreover, the reciprocal nature of these shifts suggests that an interconversion of one cell type to another may have occurred.

Animals↗
PubMed1984

Brief decreases in dopamine result in surges of prolactin secretion in monkeys.

We have reported previously that the amount of dopamine in hypophysial stalk blood was sufficient to account for tonic hypothalamic inhibition of prolactin secretion in rhesus monkeys. In the present study we determined the effect on prolactin secretion of decreases in dopamine. When hypophysial stalk-transected, estrogen-treated monkeys were infused with dopamine at a rate (0.1 microgram X kg-1 X min-1) to achieve peripheral plasma concentrations similar to those previously measured in stalk blood, their elevated serum prolactin levels fell to base line (from 47.6 +/- 4.8 to 9.7 +/- 2.7 ng/ml, mean +/- SE, n = 4). Brief interruptions of the dopamine infusions lasting for 2.5, 5.0, or 7.5 min evoked rapid increases in serum prolactin concentrations (to 27.5 +/- 8.3, 52.9 +/- 3.6, and 58.9 +/- 8.1 ng/ml, respectively, at 10 min). Maximal prolactin levels were attained within 10 to 20 min after dopamine removal and serum prolactin remained elevated for an additional 35 to 150 min. Uninterrupted dopamine infusions of stalk-transected, estrogen-treated monkeys (n = 3) led to continuously suppressed prolactin values. These results demonstrate that brief decreases in dopamine lead to major increases in prolactin secretion. Thus, if a brief decrease in hypothalamic dopamine secretion occurs after a suckling stimulus in monkeys, as it does in rats, a decrease in dopamine secretion alone may account, in part, for suckling-induced prolactin secretion in monkeys.

Animals↗
PubMed1984

A reverse hemolytic plaque assay for microscopic visualization of growth hormone release from individual cells: evidence for somatotrope heterogeneity.

Growth hormone (GH) secreting cells direct complement-mediated plaque formation (clear zones of hemolysis surrounding the somatotropes) in mixed pituitary cell cultures incubated as a monolayer with protein-A coupled ovine erythrocytes (oRBC) in the presence of GH antiserum. Plaques were maximal in number after 4 h; growth hormone-releasing hormone (GHRH) and somatostatin increased and decreased, respectively, the rate of formation of plaques and their final sizes. Approximately 30% of all pituitary cells formed GH plaques and a similar fraction stained for GH using peroxidase-antiperoxidase immunocytochemistry (ICC). The plaque areas of individual somatotropes (reflecting the amount of GH released) covered a 20-fold range from the smallest to the largest in the 3 treatment groups. Somatostatin-treated and untreated cells formed frequency distributions of plaque sizes that were unimodal. In contrast, GHRH produced a bimodal frequency distribution suggestive of a sub-population of somatotropes preferentially responsive to this secretagogue. This new assay coupled with other morphological and biochemical techniques that can be applied to single cells will permit further analysis of these sub-populations of somatotropes.

Animals↗
PubMed1984

Biphasic effects of estrogen on gonadotropin-releasing hormone-induced luteinizing hormone release in monolayer cultures of rat and monkey pituitary cells.

The negative feedback effect of estrogen on LH secretion has been difficult to demonstrate in monolayer cultures of rat pituitary cells. The purpose of the present study was to establish the experimental conditions required for manifestation of this response and, in the process, to develop models for investigating the actions of steroids on pituitary cells. Both dynamic and static incubation systems were used. For perifusion experiments, trypsin-dispersed pituitary cells from rats at random stages of the estrous cycle were attached to glass coverslips with poly-L-lysine, incubated for 48 h, and then mounted in Sykes-Moore chambers. In each of these experiments, two chambers were perifused concurrently: one with medium containing 1.8 X 10(-10) M 17 beta-estradiol and the other with medium alone. GnRH (4.2 X 10(-9) M) was coinfused for 5 min out of every hour, and samples of perifusate were collected as 5-min fractions for assay of LH. Estrogen treatment significantly (P less than 0.01) suppressed LH release in response to the first five GnRH pulses compared to the control value. The inhibition was most pronounced early in the perifusion, but had disappeared by 6 h. These results demonstrate that estradiol exerts a potent but transient inhibition of GnRH-induced LH release in monolayer cultures of rat pituitary cells. In a subsequent set of experiments, we modified a static incubation system to assess sequentially the biphasic effects of estrogen on LH release by the same group of cells. Cultures of rat pituitary cells that had been established 42 h previously were treated simultaneously for 3 h with 17 beta-estradiol (3.7 X 10(-10) M) and various concentrations of GnRH (5 X 10(-10) to 1 X 10(-7) M) to measure the inhibitory effects of the steroid on LH secretion. This experiment was repeated on the same cells after 27 h of steroid exposure to estimate the facilitory actions of estrogen on LH release. The negative feedback of estrogen was demonstrable in static cultures of rat pituitaries provided that the period of estrogen exposure and duration of incubation were brief. Moreover, the results indicate that the same groups of cells can be used on consecutive days to investigate the inhibitory and stimulatory effects of estrogen on LH secretion. Experiments with cultures of monkey pituitary cells yielded similar results. Taken together, these findings indicate that cultured pituitary cells are responsive to the biphasic actions of estradiol and demonstrate the utility of two model systems for investigating these phenomena.

Animals↗
PubMed1984

Detection of LH release from individual pituitary cells by the reverse hemolytic plaque assay: estrogen increases the fraction of gonadotropes responding to GnRH.

Gonadotropes that secrete LH were demonstrated microscopically among mixed anterior pituitary (AP) cells in culture with a reverse hemolytic plaque assay. LH released from a cell binds to adjacent RBCs bearing an LH Ab-protein A complex which results in a zone of complement-mediated hemolysis (a plaque) surrounding each gonadotrope. In untreated cultures a few, small plaques formed, but in the presence of hypothalamic GnRH (10(-7) M, a maximally stimulating dose) a 10-fold increase occurred in their number and size. Non-secretory gonadotropes were found in the pituitary of diestrous animals: 5-6% of all AP cells contained immunocytochemically detectable LH whereas only 2.5-3.0% formed plaques in the presence of GnRH (10(-7) M). Nearly all of the gonadotropes were secretory at proestrus. Estradiol treatment of diestrous cultures for 24 h increased the fraction of secretory gonadotropes to near the level observed in proestrous cultures. These results demonstrate the utility of the reverse hemolytic plaque assay for detection of LH secretion from individual gonadotropes, establish that not all cells containing LH can secrete it, and suggest that a previously unrecognized mode of estrogen action to evoke the preovulatory LH surge is mediated by increasing the fraction of secretory gonadotropes.

Animals↗
PubMed1983

Nursing induces a biphasic release of prolactin in rhesus monkeys.

The effect of nursing on serum PRL levels was studied in four conscious unrestrained rhesus monkeys (20--46 days postpartum) fitted previously with exteriorized cardiac catheters. On the day of an experiment, the infant was transferred to a separate room for 9.5 h. Blood samples for PRL assay were collected from the mother at frequent intervals for 1 h before and 3 h after returning the infant. In all instances, vigorous suckling of the nipple was observed within 10 min of reuniting mother and infant and resulted in an elevation of serum PRL from an initial value of 7.4 +/- 0.5 ng/ml (mean +/- SEM) to a peak at 90 min of 339.7 +/- 56.1 ng/ml. Analysis of secretory patterns from individual animals revealed that PRL release was biphasic. During phase I, which began within 10--50 min of returning the infants and lasted for 20--50 min, serum PRL concentrations increased at an average rate of 0.47 ng/ml . min. This was followed by phase II, lasting 20--40 min, during which serum PRL rose at a 20-fold greater rate (10.57 ng/ml . min) to sustained maximum levels. Whether this unusual pattern of PRL secretion occurs as a consequence of neuroendocrine interactions unique to this species or simply reflects the prolongation and partitioning of a secretory process common to other mammals remains to be determined.

Animals↗
PubMed1983

Detection of hormone release from individual cells in mixed populations using a reverse hemolytic plaque assay.

Prolactin (Prl) secreting cells in a mixed pituitary cell culture form microscopically-identifiable plaques (zones of hemolysis around the lactotropes) when incubated in a monolayer with staphylococcal protein-A-coated ovine erythrocytes in the presence of Prl antiserum and complement. Plaques form first at 15-30 min and are maximal in size and number at 2 h. Approximately 70% of the adenohypophyseal cells form plaques under these conditions. TRH increases, and dopamine decreases, the size and number of plaques at early times during incubation. This reverse hemolytic plaque assay probably can be used to detect any cell secretion for which an antibody is available. This technique, or a modified version of it in which sequential plaque assays are performed on identified cells--used together with immunocytochemistry, autoradiography or electron microscopy of those cells--should provide better answers to commonly asked questions about secretory systems: Do all or only a subset of cells containing the same hormone respond to a particular secretagogue? Can cells that contain two hormones release one of them preferentially?

Animals↗