Simultaneous measurement of [Ca2+]i and secretion-coupled membrane turnover, by single cell fluorescence microscopy

Shorte, Spencer; Stafford, Sarah J.V.; Collett, Valerie J.; Schofield, Jill

doi:10.1016/0143-4160(95)90059-4

Cited by 20 publications

(12 citation statements)

References 45 publications

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“…Note the difference in the type of response to E2 (slow gradual rise in Ca¥) vs. TRH (rapid spike and return to baseline). The TRH pattern is similar to that observed previously (Shorte et al 1995). In three experiments, we assessed 137 cells of which 51 (37%) responded to both hormones, 34 (25%) responded to E2 alone, 38 (28%) responded to TRH alone, and 14 (10%) either did not respond or were not categorizable.…”

Section: ------------------------------------------------------------supporting

confidence: 55%

“…We reasoned that we should address this problem with a technique which allows assessment of individual cells, because even in the F10 subclone, mER expression is dynamic, and only a percentage of the population at any given time bears significant amounts of mER on the cell surface. We also compared the response to E2 to that of a well-characterized response to the peptide TRH (Shorte et al 1995). Figure 2 shows the averaged profiles of cells demonstrating both TRH-and oestrogen-induced Ca¥ level increases.…”

Section: ------------------------------------------------------------mentioning

confidence: 99%

“…The responses to 10 nÒ E2 and 1 ìÒ thyrotropin-releasing hormone (TRH; Martin, 1983;Ohmichi et al 1994;Shorte et al 1995) are shown over a 9·5 min time course via fluorescence imaging on a Nikon Diaphot inverted microscope coupled to a dual monochrometer system via a fibreoptic cable (Photon Technology International, Monmouth Junction, NJ, USA). Images were processed using ImageMaster software (Tsien & Harootunian, 1990).…”

Section: Analysis Of Ca¥ By Whole Cell Imagingmentioning

confidence: 99%

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Membrane Oestrogen Receptors on Rat Pituitary Tumour Cells: Immuno‐Identification and Responses to Oestradiol and Xenoestrogens

Watson

Campbell

Gametchu

1999

Experimental Physiology

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summaryOur laboratory has identified plasma membrane oestrogen receptors on a GH×ÏB6 rat pituitary tumour cell line and several sublines which produce rapid (within minutes), non-genomic responses to oestrogens. Oestrogen receptors have been identified by their binding to nine different antibodies (Abs) which together recognize at least seven epitopes on the oestrogen receptor-á. GH×ÏB6ÏF10 cells, a membrane oestrogen receptor-enriched subline, elevate intracellular calcium levels in response to 10 nÒ oestradiol. Prolactin release in these cells is triggered by both 1 pÒ and 1 nÒ oestradiol and diethylstilbestrol (DES). A membrane oestrogen receptor-á immunocyto-chemical signal rapidly disappears (at 3 min) and reappears (at 12-15 min) when 1 nÒ oestradiol,

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Section: ------------------------------------------------------------supporting

confidence: 55%

Section: ------------------------------------------------------------mentioning

confidence: 99%

Section: Analysis Of Ca¥ By Whole Cell Imagingmentioning

confidence: 99%

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Membrane Oestrogen Receptors on Rat Pituitary Tumour Cells: Immuno‐Identification and Responses to Oestradiol and Xenoestrogens

Watson

Campbell

Gametchu

1999

Experimental Physiology

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“…Downstream intracellular signaling responses, such as the production of IP 1 and the activity of ERK 1/2 , which will be discussed later, can be reflected in the oscillation patterns associated with the different disease-related mutations. Although most research has focused on intracellular calcium mobilization in HEK293 cells heterologously expressing the CaSR, CaSR-induced [Ca 2+ ] i oscillations have also been found in cells with endogenously expressed CaSR, for instance in parathyroid cells [31], bovine anterior pituitary cells [32], opossum kidney (OK) cells [33] and medullary thyroid carcinoma cells [34]. Oscillations in [Ca 2+ ] i modulate not only the rate of parathyroid hormone (PTH) secretion, but also gene expression and other processes [35]–[38].…”

Section: Discussionmentioning

confidence: 99%

Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease-Associated “Toggle” Calcium-Sensing Receptor Mutations

et al. 2014

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The Ca2+-sensing receptor (CaSR) regulates Ca2+ homeostasis in the body by monitoring extracellular levels of Ca2+ ([Ca2+]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca2+]o–induced [Ca2+]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro221 and Leu173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.

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“…Furthermore, the absorption and emission wavelengths of FM1-43 (502 and 625 nm respectively) allow it to be used simultaneously with measurement of [Ca# + ] i using fura 2 (whose optimal absorption and emission wavelengths are 340\380 nm and 510 nm respectively) [21]. A dual-labelling technique with FM1-43 and fura 2 was used previously in our laboratory to study secretion from bovine anterior pituitary cells [24,25].…”

Section: Introductionmentioning

confidence: 99%

Changes in cytoplasmic calcium determine the secretory response to extracellular cations in human parathyroid cells: a confocal microscopy study using FM1-43 dye

Mihai

Lai

Schofield

et al. 2000

Biochemical Journal

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Whether activation of the calcium receptor (CaR) modulates secretory events was investigated by real-time fluorescence and confocal microscopy using fura 2 and FM1-43 fluorescent dye. Two paradigms were used : human parathyroid cells, which are stimulated by a step from a high to a low extracellular calcium concentration ([Ca# + ] ext ), and rMTC6-23 cells, a rat medullary thyroid carcinoma cell line whose secretion is stimulated by an increase in [Ca# + ] ext . Parathyroid cells were dispersed from parathyroid adenomas removed from 18 patients with primary hyperparathyroidism. In both cell types, incubation with FM1-43 (2 µM) resulted in staining of the plasma membranes, which was rapidly increased following changes in [Ca# + ] ext known to stimulate secretion. A high [Ca# + ] ext and lanthanum (La$ + ) decreased the membrane-associated FM1-43 fluorescence. Prolonged incubation (5-30 min) in the presence of FM1-43 resulted in accumulation of the dye in the cytoplasm, its granular distribution suggesting targeting of the secretory compartment. These data suggest that FM1-43 fluorescence is determined by :

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Simultaneous measurement of [Ca2+]i and secretion-coupled membrane turnover, by single cell fluorescence microscopy

Cited by 20 publications

References 45 publications

Membrane Oestrogen Receptors on Rat Pituitary Tumour Cells: Immuno‐Identification and Responses to Oestradiol and Xenoestrogens

Membrane Oestrogen Receptors on Rat Pituitary Tumour Cells: Immuno‐Identification and Responses to Oestradiol and Xenoestrogens

Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease-Associated “Toggle” Calcium-Sensing Receptor Mutations

Changes in cytoplasmic calcium determine the secretory response to extracellular cations in human parathyroid cells: a confocal microscopy study using FM1-43 dye

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