Millisecond studies of calcium-dependent exocytosis in pituitary melanptrophs: Comparison of the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen

T. D. Parsons, G. C R Ellis-Davies, Wolfhard Almers

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca]i) to trigger exocytosis. The whole cell patch clamp technique was used to monitor membrane capacitance (Cm) and to load both calcium indicator dye and photolabile chelators into rat pituitary melanotrophs prior to flash photolysis. In cells dialysed with DMN, a transient increase in [Ca]i was observed immediately after continuity between the patch pipette and the cell cytosol was achieved. This 'loading transient' reflects the release of calcium from DMN during the binding of intracellular magnesium. No such transient was seen with NPE, consistent with the negligible binding of magnesium to this chelator. Following flash photolysis of DMN or NPE, [Ca]i increased, triggering both a rapid exocytic burst and slower sustained phases of exocytosis. When flashes of the same intensity were compared, the photolysis of NPE resulted in smaller increases in [Ca]i and slower exocytic bursts than that of DMN. These findings are in accordance with the properties of the two compounds [Ellis-Davies G.C.R., Kaplan J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci USA 1994; 91: 187-191] and the calcium dependency of the exocytic burst [Thomas P., Wong J.G., Lee A.K., Almers W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 1993; 11: 93-104]. Although NPE is somewhat less effective than DMN in raising [Ca]i, this chelator promises to be a useful and interesting tool for the time-resolved study of calcium-dependent exocytosis in the presence of physiological concentrations of magnesium.

Original languageEnglish (US)
Pages (from-to)185-192
Number of pages8
JournalCell Calcium
Volume19
Issue number3
StatePublished - Mar 1996
Externally publishedYes

Fingerprint

Egtazic Acid
Exocytosis
Calcium
Photolysis
Magnesium
Melanotrophs
Chelating Agents
1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis((oxycarbonyl)methyl)-1,2-ethanediamine
Calcium Chelating Agents
Patch-Clamp Techniques
Cytosol
Coloring Agents
Neurons
Peptides
Membranes

ASJC Scopus subject areas

  • Cell Biology
  • Endocrinology

Cite this

Millisecond studies of calcium-dependent exocytosis in pituitary melanptrophs : Comparison of the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen. / Parsons, T. D.; Ellis-Davies, G. C R; Almers, Wolfhard.

In: Cell Calcium, Vol. 19, No. 3, 03.1996, p. 185-192.

Research output: Contribution to journalArticle

@article{a479681bc8bb45359ee7f198c9ff681c,
title = "Millisecond studies of calcium-dependent exocytosis in pituitary melanptrophs: Comparison of the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen",
abstract = "DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca]i) to trigger exocytosis. The whole cell patch clamp technique was used to monitor membrane capacitance (Cm) and to load both calcium indicator dye and photolabile chelators into rat pituitary melanotrophs prior to flash photolysis. In cells dialysed with DMN, a transient increase in [Ca]i was observed immediately after continuity between the patch pipette and the cell cytosol was achieved. This 'loading transient' reflects the release of calcium from DMN during the binding of intracellular magnesium. No such transient was seen with NPE, consistent with the negligible binding of magnesium to this chelator. Following flash photolysis of DMN or NPE, [Ca]i increased, triggering both a rapid exocytic burst and slower sustained phases of exocytosis. When flashes of the same intensity were compared, the photolysis of NPE resulted in smaller increases in [Ca]i and slower exocytic bursts than that of DMN. These findings are in accordance with the properties of the two compounds [Ellis-Davies G.C.R., Kaplan J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci USA 1994; 91: 187-191] and the calcium dependency of the exocytic burst [Thomas P., Wong J.G., Lee A.K., Almers W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 1993; 11: 93-104]. Although NPE is somewhat less effective than DMN in raising [Ca]i, this chelator promises to be a useful and interesting tool for the time-resolved study of calcium-dependent exocytosis in the presence of physiological concentrations of magnesium.",
author = "Parsons, {T. D.} and Ellis-Davies, {G. C R} and Wolfhard Almers",
year = "1996",
month = "3",
language = "English (US)",
volume = "19",
pages = "185--192",
journal = "Cell Calcium",
issn = "0143-4160",
publisher = "Churchill Livingstone",
number = "3",

}

TY - JOUR

T1 - Millisecond studies of calcium-dependent exocytosis in pituitary melanptrophs

T2 - Comparison of the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen

AU - Parsons, T. D.

AU - Ellis-Davies, G. C R

AU - Almers, Wolfhard

PY - 1996/3

Y1 - 1996/3

N2 - DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca]i) to trigger exocytosis. The whole cell patch clamp technique was used to monitor membrane capacitance (Cm) and to load both calcium indicator dye and photolabile chelators into rat pituitary melanotrophs prior to flash photolysis. In cells dialysed with DMN, a transient increase in [Ca]i was observed immediately after continuity between the patch pipette and the cell cytosol was achieved. This 'loading transient' reflects the release of calcium from DMN during the binding of intracellular magnesium. No such transient was seen with NPE, consistent with the negligible binding of magnesium to this chelator. Following flash photolysis of DMN or NPE, [Ca]i increased, triggering both a rapid exocytic burst and slower sustained phases of exocytosis. When flashes of the same intensity were compared, the photolysis of NPE resulted in smaller increases in [Ca]i and slower exocytic bursts than that of DMN. These findings are in accordance with the properties of the two compounds [Ellis-Davies G.C.R., Kaplan J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci USA 1994; 91: 187-191] and the calcium dependency of the exocytic burst [Thomas P., Wong J.G., Lee A.K., Almers W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 1993; 11: 93-104]. Although NPE is somewhat less effective than DMN in raising [Ca]i, this chelator promises to be a useful and interesting tool for the time-resolved study of calcium-dependent exocytosis in the presence of physiological concentrations of magnesium.

AB - DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca]i) to trigger exocytosis. The whole cell patch clamp technique was used to monitor membrane capacitance (Cm) and to load both calcium indicator dye and photolabile chelators into rat pituitary melanotrophs prior to flash photolysis. In cells dialysed with DMN, a transient increase in [Ca]i was observed immediately after continuity between the patch pipette and the cell cytosol was achieved. This 'loading transient' reflects the release of calcium from DMN during the binding of intracellular magnesium. No such transient was seen with NPE, consistent with the negligible binding of magnesium to this chelator. Following flash photolysis of DMN or NPE, [Ca]i increased, triggering both a rapid exocytic burst and slower sustained phases of exocytosis. When flashes of the same intensity were compared, the photolysis of NPE resulted in smaller increases in [Ca]i and slower exocytic bursts than that of DMN. These findings are in accordance with the properties of the two compounds [Ellis-Davies G.C.R., Kaplan J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci USA 1994; 91: 187-191] and the calcium dependency of the exocytic burst [Thomas P., Wong J.G., Lee A.K., Almers W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 1993; 11: 93-104]. Although NPE is somewhat less effective than DMN in raising [Ca]i, this chelator promises to be a useful and interesting tool for the time-resolved study of calcium-dependent exocytosis in the presence of physiological concentrations of magnesium.

UR - http://www.scopus.com/inward/record.url?scp=0030006127&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030006127&partnerID=8YFLogxK

M3 - Article

C2 - 8732258

AN - SCOPUS:0030006127

VL - 19

SP - 185

EP - 192

JO - Cell Calcium

JF - Cell Calcium

SN - 0143-4160

IS - 3

ER -