Prolactin Cell Subpopulations Separated on Discontinuous Percoll Gradient: An Immunocytochemical, Biochemical, and Physiological Characterization*

Velkeniers, B.; Hooghe‐Peters, Elisabeth L.; Hooghe, Robert; Belayew, Alexandra; Smets, G.; Claeys, Annelies; Robberecht, Patrick; Vanhaelst, L.

doi:10.1210/endo-123-3-1619

Cited by 60 publications

(28 citation statements)

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“…The cell suspension obtained was filtered through nylon gauze, washed and centrifuged. Cells were grown for 3 days in uncoated Petri dishes in Dulbecco's Modified Eagle's Medium (DMEM) as previously reported (27). Cells were harvested, washed once with DMEM and immediately extracted as follows: they were taken up in the appropriate buffer, lysed by twice freezing and thawing, centrifuged for 30 min at 12,000 x g, and the lysate supernatants either immediately assayed or stored at -60 "C.…”

Section: Methodsmentioning

confidence: 99%

Further Characterization of Rat 26,000 Prolactin as a Glycoprotein with Essentially O‐Linked Carbohydrate Chains

Bollengier

Hooghe

Velkeniers

et al. 1991

J Neuroendocrinology

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Laboratorium voor Farrnacologie, Faculteit Geneeskunde en Farrnacie, Vrije Universiteit Brussel (V U B ) > Brussel, Belgium tDepartement Radioprotectie, Studiecentrum voor Kernenergie (S C K ), Mol, Belgium Key words: rat prolactin, glycoenzyme hydrolysis, lectin affinity chromatography, carbohydrate chain Abstract In the rat two major molecular variants of prolactin a r e recorded i.e. 23,000 M, and glycosylated 26,000 M,. In order to further characterize the glycosylated 26,000 rat prolactin molecular variant, rat pituitary cell lysates were digested with several glycoenzymes and the digestion products submitted to sodium dodecyl sulphate polyacrylarnide gel electrophoresis and subsequent immunoblotting. The results were as follows: treatment with 1) neuraminidase, specific for sialic acid, yielded an M, decrease of the glycosidic variant from 26,000 to 24,500, 23,800, 23,000 and 22,000; 2) endo-cr-N-acetylgalactosaminidase, which releases the disaccharide Gal (p 1-3) GalNac from 0-glycans, split 26,000 rat prolactin into a doublet of M, 26,000 to 25,500; and 3) mixed exoglycosidases from Turbo cornutus caused a gradual M, shift from 26,000 to 23,000.Affinity chromatography on wheat germ agglutinin Sepharose 6MB and soybean agglutinin agarose of rat pituitary homogenates and competitive inhibition tests showed that glycosylated rat prolactin has distinct affinity for these lectins.From the experimental data it is proposed that glycosylated rat prolactin is 0-linked through threonine by the disaccharide Gal (p 1-3) GalNac and possesses at least GalNac, andlor Gal and sialyl residues.Recently we identified 26,000 rat prolactin (rPRL) as a presumably 0-linked glycoprotein (1). The function of glycosylated rPRL is not yet understood, nevertheless one can speculate upon possible biological roles. In review articles by Schachter (2) and Rademacher et al. (3), it is shown that the carbohydrates of glycoproteins contribute to stabilization and secretion of proteins, to recognition of receptormediated endocytosis and are required for biological activity.Hattori et al. (4) demonstrated that the heterogeneity of rat lutotropin, a basic glycoprotein hormone, depends on sialic acid moiety as in acidic glycoproteins like human chorionic gonadotropin ( 5 ) and follicle-stimulating hormone (6), and they concluded 1 hat the number of sialic acid is responsible for the biological ilotency of the hormone. Deglycosylation of human chorionic :ionadotropin (7) and follicle-stimulating hormone (8) was consist-'. ntly associated with high affinity binding to the receptor in the ' ' m e that deglycosylated glycohormones, although biologically Illactive, were potent competitive inhibitors of binding to the 1 i ttive hormone.Only the determination of the structure-function relationship 26,000 rPRL will answer the question whether rPRL has a ~J'eCifiC activity by means of its carbohydrate moiety.In order to gain further insight into the nature of the glycosidic bond and the processing of 26,000 rPRL, we recently cultured pituitary cells ...

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Section: Methodsmentioning

confidence: 99%

Further Characterization of Rat 26,000 Prolactin as a Glycoprotein with Essentially O‐Linked Carbohydrate Chains

Bollengier

Hooghe

Velkeniers

et al. 1991

J Neuroendocrinology

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“…Such a possibility has already been proposed by other authors [28][29][30][31]: it reflects the complexity of regulatory mechanisms which can only be thoroughly investi gated by single cell studies.…”

Section: Discussionmentioning

confidence: 99%

Plateau Potentials Recorded from Lactating Rat Enriched Lactotroph Cells Are Triggered by Thyrotropin Releasing Hormone and Shortened by Dopamine

Israël¹,

Kukstas²,

Vincent³

1990

Neuroendocrinology

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Enrichment of dispersed pituitary cells from normal lactating rats on a continuous BSA gradient permitted the isolation of two prolactin cell populations – light and heavy. Hormone release studies indicated that spontaneous prolactin (PRL) release of the heavy fraction cells was particularly sensitive to TRH stimulation (mean of 450%) and that this effect of TRH was totally inhibited by Ca²⁺ channel blockers (Co²⁺, Cd²⁺, Ni²⁺ and Mn²⁺). For this reason, the electrophysiological response to TRHwas investigated on heavy fraction cells. Experiments performed on 264 cells after 4–12 days in culture showed that these cells could be divided into two groups. The first group, called high resting potential (HRP) cells, constituted 73% of the total and was characterized by a mean resting potential of –60 mV and a mean input membrane resistance of 700 MΩ, and these cells displayed plateau potentials, which were triggered by application of brief (2 s), large (1 nA) depolarizing or hyperpolarizing current steps. The plateaux were characterized by a sustained depolarization at a potential near –20 mV and they were concomitant with an increase of the membrane conductance. The repolarization consisted of a slight, gradual hyperpolarization followed by a rapid return to the resting potential. The second group of cells (the remaining 27%; called low resting potential or LRP cells) was characterized by a mean resting potential of –45 mV and a mean input membrane resistance of 250 MΩ. These cells were excitable, 30% of them displaying spontaneous activity but never showing plateaux. Electrophysiological experiments showed that the majority of the HRP cells (99%) responded to TRH but were insensitive to dopamine (DA) ejections (up to 10^–6M) at resting potential. Ejection of TRH onto HRP cells induced a slow depolarization (10–15 mV) concomitant with a decrease of the membrane resistance. This led the cell membrane potential to a critical value (≈–50 mV), at which the plateau response was triggered. The plateau lasted for about 10 s from potentials greater than –100 mV, and could reach 10 min from holding potentials close to the resting potential. The amplitude of the plateau varied according to the holding potential and the reversal potential was found to be -20 mV. Local application of tetraethylammonium chloride (TEA, 30 mM) only slightly affected the amplitude of the plateaux but they were shortened or totally blocked by Co²⁺, Cd²⁺ and Ni²⁺. Contrary to is lack of effect on these cells at their resting potential, the ejection of DA onto cells showing plateau activity shortened or totally abolished the plateau. This effect was mimicked by the dopaminergic agonist bromocriptine. On the other hand, 60% of the LRP cells were insensitive to TRH (10^–6M) but showed a hyperpolarization after DA application (40% were insensitive either to DA or to TRH). We conclude that two subpopulations of PRL cells may exist, one which, at resti...

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“…These methods have exploited the differences in density and size of the individual cell types after enzymic dispersion to provide a means for their separation. Such methods commonly included centrifugal elutriation [10][11][12][13][14][15] or sedimentation at unit gravity [16][17][18][19][20][21], Flow cytometric analysis and sorting allows simultaneous purification and depletion of differing anterior pituitary cell types [5,6], This is a slow and labor-intensive process but has the advantage of providing high purity of the entire cell type, and much additional information on the morphological charac teristics of the separated cells.…”

Section: Discussionmentioning

confidence: 99%

Magnetic Bead Separation of Anterior Pituitary Cells

Wynick¹,

Bloom²

1990

Neuroendocrinology

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The use of magnetic beads coated with anti-IgG antibodies should allow simultaneous purification and depletion of differing anterior pituitary cell types labelled with anti-hormone antibodies. This technique would be expected to give very similar results to fluorescence-activated cell sorting (FACS). Magnetic bead separation of dispersed, labelled anterior pituitary cells is cheap, easy and quick to perform (time from the end of anti-hormone antibody labelling to completion of purification is approximately 30 min) and the resulting cells are viable for at least 24 hours after purification. While the cell recovery for beads and FACS, 94% (SEM ± 4.4) vs. 89% (SEM ± 3.9) and purity of 88% (SEM ± 2.2) vs. 96.7% (SEM ± 1.7) for lactotrophs and purity of 87% (SEM ± 1.9) vs. 98% (SEM ± 1) for somatotrophs are similar, the results for depletion by the magnetic bead separation method are disappointing, only 30–40% of the labelled lactotrophs or somatotrophs cells bind to the beads and thus only a sub-population of cells may be purified by this method. These results are explicable on the basis of the sensitivity of the two techniques. Pituitary cells co-incubated with two specific anti-prolactin antibodies (one raised in rabbit and one in sheep) demonstrate that removal by Dynal magnetic beads (coated with rabbit IgG antibody) of those prolactin molecules bound to the rabbit anti-prolactin antibody also removed those prolactin molecules bound to a sheep anti-prolactin antibody. In contrast, co-incubating cells with the rabbit anti-prolactin antibody and a sheep anti-growth hormone antibody did not remove growth hormone labelling when the prolactin bound to the beads was removed. We hypothesise that during exocytosis at 4 °C, the hormone core of the secretory granule is transiently trapped within the lipid bi-layer of the surface membrane prior to discharge and it is at this point that the intact hormone core is labelled with the anti-hormone antisera. Removal of the prolactin core by the bead separation technique would simultaneously remove the prolactin bound to the sheep anti-prolactin antibody yet in no way affect growth hormone which might be present on a neighbouring cell.

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Prolactin Cell Subpopulations Separated on Discontinuous Percoll Gradient: An Immunocytochemical, Biochemical, and Physiological Characterization*

Cited by 60 publications

References 0 publications

Further Characterization of Rat 26,000 Prolactin as a Glycoprotein with Essentially O‐Linked Carbohydrate Chains

Further Characterization of Rat 26,000 Prolactin as a Glycoprotein with Essentially O‐Linked Carbohydrate Chains

Plateau Potentials Recorded from Lactating Rat Enriched Lactotroph Cells Are Triggered by Thyrotropin Releasing Hormone and Shortened by Dopamine

Magnetic Bead Separation of Anterior Pituitary Cells

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