Superfusion and static culture techniques for measurement of rapid changes in prolactin secretion

Escalera, Gonzálo Martínez de la; Swearingen, Karen C.; Weiner, Richard I.

doi:10.1016/0076-6879(89)68018-4

Cited by 19 publications

(16 citation statements)

References 16 publications

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“…During the second 30-min incubation period, part of the cells preincubated in the absence of DA were challenged with DA, while part of the cells preincubated with DA were incubated in medium without DA. Medium changes were performed by transferring the cell-coated coverslips to prefilled Petri dishes, minimizing the mechanical disturbance of the cells, as previously described [18]. The release of PRL into the medium was assessed from sextuplicate wells, after removing detached cells by microcentrifugation at 15,000 g for 15 min and storing the samples at –20°C until subjected to radioimmunoassay (RIA).…”

Section: Methodsmentioning

confidence: 99%

“…Cells frozen in 10% dimethylsulfoxide medium plus serum were periodically thawed (every 6–8 weeks) and introduced as the stock line. In addition, anterior pituitaries of female Sprague-Dawley rats bilaterally ovariectomized and implanted with 1-cm estradiol-filled Silastic capsules (Dow Corning, Midland, Mich., USA; 0.125 inch od; 0.062 inch id) for 14 days, were dispersed and cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% calf serum, 100 U/ml penicillin-streptomycin, 25 µg/ml fungizone and 100 µg/ml gentamycin as described previously [18]. Cells were plated on 12-mm Matrigel-coated plastic coverslips (Thermanox, Miles Laboratories) placed in 15-mm plastic Petri dishes, and maintained at 37°C in a water-saturated atmosphere of 95% O 2 and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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Potentiation of Prolactin Secretion following Lactotrope Escape from Dopamine Action

et al. 1999

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Hypothalamic dopamine (DA) tonically inhibits prolactin (PRL) release from the anterior pituitary gland. Transient escapes from this DA tone elicit a pronounced potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). Previous evidence has suggested that modulation of Ca²⁺ channels can be involved in this potentiation. With a lactotropic cell line (GH₄C₁) expressing human D₂-DA receptors, we tested the hypothesis that a brief escape from the tonic inhibitory action of DA triggers a facilitation of Ca²⁺ influx through Ca²⁺ channels. We initially found that in these cells, DA effectively and reversibly inhibited PRL secretion, and reversibly enhanced an inwardly rectifying K⁺ current. The effects of DA administration and withdrawal on Ca²⁺ currents were examined using the patch-clamp technique in the whole-cell configuration and Ba²⁺ as a divalent charge carrier through Ca²⁺ channels. Macroscopic Ba²⁺ currents were significantly decreased by short term (1–10 min) applications of DA (500 nM), which further declined following 24 h of constant exposure to DA. After DA removal, a biphasic facilitation of the density of Ba²⁺ currents was observed. An initial 2-fold enhancement of conductance was detected between 10 and 40 min, followed by a second facilitation of the same magnitude observed 24 h after DA withdrawal. The present results directly demonstrate that dissociation of DA from D₂-receptors expressed in GH₄C₁ lactotrope cells causes an increase of high-voltage-activated Ca²⁺ channel function, which may play an important role in the cross-talking amplification of endocrine cascades such as that involved in the TRH-induced PRL-release potentiating action of DA withdrawal.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Potentiation of Prolactin Secretion following Lactotrope Escape from Dopamine Action

et al. 1999

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“…Cells were superfused in Sykes-Moore chambers (Bellco Glass) consisting of two coverslips separated by a rubber gasket. The coverslips were compressed against the gasket to form a chamber with a volume of 200 Al, which was superfused with medium (13).…”

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confidence: 99%

Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line.

Escalera

Choi

Weiner

1992

Proc. Natl. Acad. Sci. U.S.A.

235

130

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The immortalized neuronal cell line GT1-1 was used to investigate the endogenous pattern of GnRH release. The GT1-1 cell line was derived from a GnRHsecreting tumor in a transgenic mouse induced by genetically targeted expression of the potent simian virus 40 oncogene encoding tumor antigen. Cells attached to coverslips were superfused in Sykes-Moore chambers with Locke's medium, Ca2+-free Locke's medium, or Opti-MEM (another defined medium) for 2 hr, and samples were collected at 4-min intervals. Release of GnRH in 17 of 18 superfusion chambers was seen to be pulsatile when data were analyzed by cluster analysis. No significant differences were observed whether only one or both of the coverslips forming the chamber were coated with cells. Pulses exhibited a mean interpulse interval of 25.8 ± 1.5 min, a mean duration of 18.8 ± 1.4 min, and a mean amplitude of 150.5 + 6.0% above preceding nadir.

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“…Cells frozen in 10% dimethylsulfoxide medium plus serum were periodically thawed (every 6–8 weeks) and introduced as the stock line. In addition, anterior pituitaries from female Sprague-Dawley rats bilaterally ovariectomized and implanted with 1-cm estradiol-filled Silastic capsules (Dow Corning, Midland, Mich., USA; 0.125 inch od; 0.062 inch id) for 14 days, were enzymatically dispersed and cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% calf serum, 100 U/ml penicillin-streptomycin, 25 µg/ml fungizone and 100 µg/ml gentamycin as described previously [21]. Cells were plated on 12-mm Matrigel-coated plastic coverslips (Thermanox, Miles Laboratories) placed in 15-mm plastic Petri dishes, and maintained at 37°C in a water-saturated atmosphere of 95% O 2 and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Potentiation of Prolactin Secretion following Lactotrope Escape from Dopamine Action

et al. 1999

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Modulation of Ca²⁺ channels has been shown to alter cellular functions. It can play an important role in the amplification of signals in the endocrine system, including the pleiotropically regulated pituitary lactotropes. Prolactin (PRL) secretion is tonically inhibited by dopamine (DA), the escape from which triggers acute episodes of hormone secretion. The magnitude of these episodes is explained by a potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). While the mechanisms of this potentiation are not fully understood, it is known to be mimicked by the dihydropyridine, L-type Ca²⁺ channel agonist Bay K 8644 and blocked by nifedipine and methoxyverapamil. The potentiation is also blocked by inhibitors of cyclic AMP-dependent protein kinase and protein kinase C. We recently described that the escape from tonic actions of DA results in increased macroscopic Ca²⁺ currents in GH₄C₁ lactotropic clonal cells transfected with a cDNA encoding the long form of the human D₂-DA receptor. Here we show that the withdrawal from DA potentiates the PRL-releasing action of TRH in GH₄C₁/D₂-DAR cells to the same extent as in enriched lactotropes in primary culture. In both experimental models a low density of dihydropyridine receptors was shown by (+)-[³H]PN200-110 binding. Photoaffinity labelling with the dihydropyridine [³H]azidopine revealed a protein consistent with the α₁ subunit of L-type Ca²⁺ channels of 165–170 kDa. In both experimental models, the facilitation of TRH action triggered by the escape from DA was correlated with an enhanced rate of phosphorylation of this putative α₁ subunit, the nature of which was further supported by immunoprecipitation with selective antibodies directed against the α_1C and α_1D subunit of voltage-gated calcium channels. We propose that PKA- and PKC-dependent phosphorylation of the α₁ subunit of high voltage activated, L-type Ca²⁺ channels is responsible for the facilitation of Ca²⁺ currents in lactotropes, and hence for the potentiation of secretagogue-mediated PRL secretion. Thus, phosphorylation of Ca²⁺ channels in endocrine cells may be a mechanism for the regulation of various functions including amplification of hormone secretion.

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Superfusion and static culture techniques for measurement of rapid changes in prolactin secretion

Cited by 19 publications

References 16 publications

Potentiation of Prolactin Secretion following Lactotrope Escape from Dopamine Action

Potentiation of Prolactin Secretion following Lactotrope Escape from Dopamine Action

Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line.

Potentiation of Prolactin Secretion following Lactotrope Escape from Dopamine Action

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