Single Cell Monitoring of Cytosolic Calcium Reveals Subtypes of Rat Lactotrophs with Distinct Responses to Dopamine and Thyrotropin-Releasing Hormone*

Winiger, Benoît P.; Wuarin, François; Zahnd, G; Wollheim, Claes B.; Schlegel, Werner

doi:10.1210/endo-121-6-2222

Cited by 68 publications

(32 citation statements)

References 36 publications

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“…Anterior pituitary glands were removed from adult male Wistar rats (weight between 200 and 250 g) and dispersed as described elsewhere (17). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% horse serum and 2.5% foetal calf serum at 37 "C under a water-saturated atmosphere of 5% CO,, 95% air.…”

Section: Methodsmentioning

confidence: 99%

“…Intracellular quinacrine and fura-2 fluorescence were monitored on a setup described in detail previously (17,42). on an inverted microscope (Nikon) in the epifluorescence mode.…”

Section: Methodsmentioning

confidence: 99%

“…(See text for the choice of the two wavelengths). Fluorescence of single cells was monitored through a pinhole diaphragm slightly larger than the cells and an interference filter (505 nm), photons being sampled in separate channels for the two excitation wavelengths with a time constant of 300 ms. During [Ca"], monitoring, cells were kept at 37 "C and continually superfused with Hepes buffered (pH 7.4) Krebs-Ringer bicarbonate buffer, (KRBH) (17,42) with 0.1% bovine serum albumin (BSA).…”

Section: Methodsmentioning

confidence: 99%

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Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

Chiavaroli

Vacher

Vecsey

et al. 1991

J Neuroendocrinology

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Quinacrine, a fluorescent basic molecule, accumulates in secretory granules of pituitary cells, as was revealed by its colocalization with immunoreactive prolactin. Thus quinacrine fluorescence may be used to monitor secretory activity at the single cell level. Rat pituitary cells in primary culture were loaded with quinacrine and stimulated with physiological secretagogues, such as thyrotrophin-releasing hormone or bradykinin, which induced a multiphasic lowering of fluorescence, corresponding to the loss of quinacrine contained in exocytosed granules. Quinacrine was further used in combination with the fluorescent calcium probe fura-2, in order to monitor simultaneously exocytosis and variations in the cytosolic free calcium concentration,

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Section: Methodsmentioning

confidence: 99%

“…Intracellular quinacrine and fura-2 fluorescence were monitored on a setup described in detail previously (17,42). on an inverted microscope (Nikon) in the epifluorescence mode.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

Chiavaroli

Vacher

Vecsey

et al. 1991

J Neuroendocrinology

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“…6) (2). However, the precise source of the Ca2-+ involved in this response is somewhat controversial (2,18,19 (15,20).…”

Section: Methodsmentioning

confidence: 99%

Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx.

Shangold

Murphy

Miller

1988

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

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We examIned the effects of gonadotropinreleasing hormone (GnRH) (10). We now report the use of these techniques to show that GnRH produces Ca2" transients in identified gonadotropes with a very characteristic and complex time course. The role of these transients in the control of gonadotropin release is discussed. MATERIALS AND METHODSCell Culture. Female 35-day-old Sprague-Dawley rats were decapitated, their pituitaries were removed, and adenohypophyses were dissected from neurohypophyses. Anterior pituitaries were then enzymatically dispersed for 20 min with 0.25% trypsin (bovine, type XII-S, Sigma) in a metabolic shaker at 370C. Cells were then filtered through organza cloth (Tetko, Elmsford, NY), diluted to 50 ml in medium 199 with Hanks' salts and L-glutamine (GIBCO), and centrifuged at 350 x g for 10 min. The supernatant was removed, and cells were resuspended by trituration through a Pasteur pipette in 5 ml ofmedium and then were plated on sterile, poly(L-lysine)-coated (10 ,ug/ml, Sigma) etched-grid coverslips (Bellco). The cell density was z'2.5 x 105 cells per coverslip. The cell yield was z4 x 105 cells per rat. Cells were incubated for 2 days in 60-mm Falcon culture dishes, with medium 199 containing 10% (vol/vol) heat-inactivated horse serum (GIBCO) and 2.5% (vol/vol) fetal bovine serum (GIBCO) and supplemented with penicillin (5 units/ml) and streptomycin (50 pg/ml), in a 5% C02/95% air atmosphere in a water-jacketed incubator at 370C.Identification of Isolated Gonadotropes. Sheep erythrocytes (Colorado Serum, Denver) were washed four times in 0.9o NaCl and conjugated to protein A (0.5 mg/ml, Sigma) with CrCl2 (0.2 mg/ml). After further washing, cells were then mixed with LH antiserum (1:5), guinea pig complement (1:10, GIBCO), and GnRH (100 nM, Peninsula Laboratories, San Carlos, CA).

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“…Using this method, subgroups of lactotrophs were isolated on the basis of their size and secretory granule content. Today, several methods give further evidence for the functional heterogeneity of lactotrophs, including: (1) immunocytochemical studies (Saint John, Dufy-Barbe & Barker, 1986); (2) pulse-chase experiments with isotope labelling, used to compare the release of newly synthesized and older PRL (Walker & Farquhar, 1980;Morin, Rosenbaum & Tixier-Vidal, 1984); (3) the reverse haemolytic plaque assay (RHPA) method for the detection of hormone secretion by individual cells (Boockfor, Hoeffler & Frawley, 1986;Frawley & Clark, 1986;Luque et al 1986;Boockfor & Frawley, 1987); (4) radioimmunoassay combined with in situ hybridization (Velkeniers, Hooghe-Peters, Hooghe, Belayew, Smets, Claeys, Robberecht & Vanhaelst, 1988); (5) the use of fluorescent intracellular calcium probes such as Fura-2, which allows measurement of [Caa2+]i (Malgaroli et al 1987;Winiger et al 1987;Lewis et al 1988).…”

Section: Two Functional States In Rat Lactotrophsmentioning

confidence: 99%

Physiological characterization of two functional states in subpopulations of prolactin cells from lactating rats.

Lledo

Guérineau

Mollard

et al. 1991

The Journal of Physiology

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SUMMARY1. Lactotroph cells from lactating female rat pituitary glands were dissociated, separated and enriched on a continuous gradient of bovine serum albumin at unit gravity. Two lactotroph subpopulations were observed in the light (F(3-5)) and the heavy (F(7-9)) fractions of the gradient. Both populations were maintained for at least 6 days in culture before experiments were performed.2. Patch-clamp recordings, in the whole-cell mode, were performed on both lactotroph subpopulations in order to measure passive membrane properties and Ca2" currents. Resting membrane potential as well as membrane capacitance values were found to be lower in light fraction cells. The two components of Ca2+ currents, called fast and slow deactivating (FD and SD) currents, were present with different proportions in each subpopulation; the ratio of current amplitudes, SD/FD, was 2-42 + 0'41 (n = 18) in light fraction cells and 1 17 + 0-27 (n = 17) in heavy fraction cells (P < 0 02).3. Reverse haemolytic plaque assay showed that in the light and heavy fractions, 68 and 47 % of the lactotroph cells, respectively, were secreting. Population analysis of the plaque areas revealed a bimodal frequency distribution of plaque sizes consisting of small (1500 jtm2) and large plaques (3995 /tm2). A majority of light fraction cells produced large plaques whereas most of the heavy fraction cells produced small plaques.4. Perifusion experiments performed on enriched prolactin cells showed that (1) basal prolactin (PRL) release was higher in light fraction than in heavy fraction cells, (2) the dopamine (10-8 M)-induced inhibition of PRL release was greater in light fraction cells (86 + 15 %) than in heavy fraction cells (41 + 21 %), and (3) the thyrotrophin-releasing hormone (TRH, 10-8 M)-induced increase of PRL release was 150 + 60 % in light fraction versus 330+82 % in heavy fraction cells.5. Current-clamp recordings were performed using the intracellular technique.* To whom all reprint requests should be sent. MS 8802 P. -M. LLEDO AND OTHERSLactotrophs were categorized according to their electrophysiological response following application of dopamine or TRH (both 10-8 M). In the light fractions, the majority of the cells tested were hyperpolarized by dopamine (68%), whereas only 7 % were depolarized by TRH application. In the heavy fractions, most of the cells (63%) responded to TRH application, while only 13% were dopamine sensitive. the activity of the lactotroph subpopulation with a high basal release, while TRH appears effective on the subpopulation characterized by a low basal release. We discuss the possible existence of a cell cycle and the way in which it might be regulated.

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Single Cell Monitoring of Cytosolic Calcium Reveals Subtypes of Rat Lactotrophs with Distinct Responses to Dopamine and Thyrotropin-Releasing Hormone*

Cited by 68 publications

References 36 publications

Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx.

Physiological characterization of two functional states in subpopulations of prolactin cells from lactating rats.

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