Secretion from corticotropes after avidin-fluorescein stains for biotinylated ligands (CRF or AVP)

Childs, Gwen V.; Unabia, Geda; Burke, Jo Ann; Marchetti, Carla

doi:10.1152/ajpendo.1987.252.3.e347

Cited by 13 publications

(9 citation statements)

References 11 publications

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“…The magnitude of the increase seen at PRO-AM was not as great when visualized with avidin-fluorescein. This could be the result of a rapid internalization of the lig and-fluorescein complex into vesicles with a pH low en ough to quench the fluorescence [8]. The 2-to 3-fold in crease in GnRH-receptive cells between D-II-AM and PRO-AM was of the same magnitude as that seen in GnRH receptors determined by radioreceptor assays.…”

Section: Discussionmentioning

confidence: 81%

Changes in the Number of GnRH-Receptive Cells during the Rat Estrous Cycle: Biphasic Effects of Estradiol

Lloyd¹,

Childs²

1988

Neuroendocrinology

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The number of gonadotropin-releasing hormone (GnRH) receptors is known to vary throughout the estrous cycle and in other endocrine states in the rat. These changes in receptors parallel closely the concentrations of serum estradiol during the cycle. In the present study, we used two different cytochemical techniques to determine if changes in GnRH receptors represented alterations in the number of GnRH-receptive cells. Furthermore, we tested the effects of estradiol pretreatment on this phenomenon. Dispersed pituitary monolayers taken at different stages of the cycle were stimulated for 3 min with 1 nM biotinylated [D-lys⁶]-GnRH (bio-GnRH) which was localized using the avidin-biotin-peroxidase complex (ABC) technique and a black peroxidase substrate. Parallel groups were stained, while living, with an avidin-fluorescein conjugate. Some monolayers were pretreated with physiological concentrations of estradiol benzoate (1 nM – 1 pM) prior to bio-GnRH exposure and ABC stains. The resulting stains demonstrated that the percentage of bio-GnRH-receptive cells was 2–3 times greater at 10.00 h proestrus (20.2 ± 4%) when compared to the same time in estrus (8.7 ± 2%), diestrus I (5.2 ± 0.5%), diestrus II (7.6 ± 1%), and at 17.00 h diestrus II (11.4 ± 0.9%) and proestrus (7.4 ± 0.8%). These data correlated well with those obtained from living gonadotropes stained with avidin-fluorescein. Estradiol exerted a biphasic effect dependent upon the stage of the cycle at which the cells were taken.Estradiol (1 pM) treatment of monolayers taken at 10.00 h diestrus I and II increased the percentage of GnRH-receptive cells to that normally observed at 17.00 h diestrus II (11.01 ± 1.5 and 11.9 ± 1%, respectively). However, the same concentration of steroid reduced the number of GnRH-receptive cells taken at 10.00 h proestrus to levels normally observed at diestrus I (6.4 ± 0.7%). These results indicate that changes in GnRH receptors during the estrous cycle represent fluctuations in the percentage of GnRH-bound cells, and that circulating levels of estradiol can mediate this change in number. It is suggested that the recruitment of GnRH-receptive cells, in conjunction with increases in both total and multihormonal gonadotropes, prepares these cells to support the preovulatory gonadotropin surge.

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Section: Discussionmentioning

confidence: 81%

Changes in the Number of GnRH-Receptive Cells during the Rat Estrous Cycle: Biphasic Effects of Estradiol

Lloyd¹,

Childs²

1988

Neuroendocrinology

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“…Furthermore, the potentiation by AVP can be mimicked by its second messengers (12). These data suggest that some pituitary cells may have receptor levels below thresholds for detection in our cytochemical assays.…”

Section: Discussionmentioning

confidence: 68%

“…For example, not all corticotropes or thyrotropes bind AVP (18). Furthermore, not all corticotropes are sensitive to glucocorticoid inhibition or inhibitors of ion channels (11,12,19), This may reflect a maturational state of the cell. Alternatively, the individual pituitary cell types may be diverse and exhibit subtypes.…”

Section: Discussionmentioning

confidence: 99%

“…Bio-CRH or bio-AVP was tested on 3-4 day cultures of mixed pituitary cells from male rats (11,12,18,52). Diluent was as described above, only M ascorbic acid was added as an antioxidant and 100 KIU aprotinin was added as a protease inhibitor.…”

Section: Tests Of the Potency Of The Biotinylated Analogsmentioning

confidence: 99%

“…Soon after, we developed biotinylated analogs of CRH and arginine vasopressin (AVP) (11,12,18,52). The purpose of this report is to review progress in the area of identification of target binding sites for these neuropeptides.…”

Section: Introductionmentioning

confidence: 99%

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Hypothalamic regulatory peptides and their receptors: Cytochemical studies of their role in regulation at the adenohypophyseal level

Childs

Westlund

Tibolt

et al. 1991

J. Elec. Microsc. Tech.

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Hypothalamic regulatory peptides bind to specific receptors on target cells in the pituitary and control secretion. They in turn can be regulated at the pituitary level by steroid and peptide modulators. Affinity cytochemical techniques are important tools for the identification of specific target binding sites for these regulatory peptides. This presentation reviews the work in which potent, biotinylated ligands of gonadotropin releasing hormone (bio-GnRH), corticotropin releasing hormone (bio-CRH), and arginine vasopressin (bio-AVP) were applied to study the target cell responses. Bio-GnRH, bio-CRH, and bio-AVP bind to membrane receptors on specific anterior pituitary cells. Dual labeling for either gonadotropin or adrenocorticotropin (ACTH) antigens further identified the target cells. After 1-3 minutes, the label was in patches or capped on the surface. After 3 minutes, it was internalized in small vesicles and sent to receptosomes and vacuoles in the Golgi complex. Eventually the biotinylated peptides, or a metabolite, was found in the lysosomes (multivesicular bodies) and a subpopulation of secretory granules. The route and rate of uptake was similar to that described for the classical receptor-mediated endocytosis process. In contrast, intermediate lobe corticotropes internalized the bio-CRH in less than 1 minute. The route through the Golgi complex appeared to be bypassed. Instead the labeled peptide was in vesicles, on the membranes of scattered vacuoles, and in multivesicular bodies. Modulation of ligand binding by steroids showed that changes in receptor numbers correlated with changes in the number of cells that bound the ligand. In male rats, dihydrotestosterone reduced the percentage of GnRH-bound cells by 50%. Most of the reduction appeared in cells that stored luteinizing hormone (LH) antigens. In diestrous female rats, estradiol increased the percentage of bio-GnRH-bound cells. However, the steroid decreased the percentage of GnRH-bound cells in cells from proestrous rats. Glucocorticoids decreased the percentage of CRH-bound corticotropes in as little as 10 minutes. Potentiation of secretion by these ligands was correlated with increases in the percentage of ligand-bound cells. AVP pretreatment of corticotropes increased the percentage of cells that bound bio-CRH. It also increased the rate of receptor-mediated endocytosis of CRH and changed the route so that the Golgi complex was bypassed. This effect could be mimicked by activation of its second messengers (calcium and protein kinase C). Similarly, CRH pretreatment increased the percentage of corticotropes that bound AVP. Thyrotropin releasing hormone (TRH) pretreatment also increased the percentage of thyrotropes that bound AVP.(ABSTRACT TRUNCATED AT 400 WORDS)

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The role of intracellular messengers in adrenocorticotropin secretion in vitro

King

Baertschi

1990

Experientia

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Adrenocorticotropin (ACTH), an opiomelanocortin peptide, is secreted from anterior pituitary corticotrophs upon stimulation with corticotropin-releasing hormone (CRH), arginine vasopressin (AVP) and several other neuropeptides. CRH, the most potent secretagogue of ACTH, stimulates ACTH secretion and biosynthesis by increasing the production of cyclic adenosine 3',5'-monophosphate (cAMP) within corticotrophs. AVP, which is a weak secretagogue of ACTH but strongly potentiates CRH-stimulated ACTH secretion, operates through the phosphatidylinositol (PI) transduction pathway. Both CRH and AVP increase cytosolic free [Ca2+] within normal corticotrophs indicating a role for Ca2+ in ACTH secretion. Glucocorticoids inhibit ACTH synthesis by suppressing transcription of the proopiomelanocortin (POMC) gene and attenuate ACTH release by decreasing cAMP accumulation stimulated by CRH. This review focuses on the roles of these intracellular messengers in ACTH secretion from normal anterior pituitary cells in vitro, and discusses the possible interactions between the cAMP, calcium and PI transduction pathways. Future areas of research are suggested such as identification of protein substrates of cAMP-dependent and Ca2(+)-dependent kinases within normal corticotrophs and evaluation of their role in ACTH biosynthesis and secretion.

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Secretion from corticotropes after avidin-fluorescein stains for biotinylated ligands (CRF or AVP)

Cited by 13 publications

References 11 publications

Changes in the Number of GnRH-Receptive Cells during the Rat Estrous Cycle: Biphasic Effects of Estradiol

Changes in the Number of GnRH-Receptive Cells during the Rat Estrous Cycle: Biphasic Effects of Estradiol

Hypothalamic regulatory peptides and their receptors: Cytochemical studies of their role in regulation at the adenohypophyseal level

The role of intracellular messengers in adrenocorticotropin secretion in vitro

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