Spatial dynamics of intracellular calcium in agonist-stimulated vascular smooth muscle cells

Neylon, Craig B.; Hoyland, John S.; Mason, William T.; Irvine, Robin F.

doi:10.1152/ajpcell.1990.259.4.c675

Cited by 102 publications

(38 citation statements)

References 6 publications

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“…More recent studies using fluorescent dextrans arrive at the same conclusion and show that dextrans with molecular weights up to 17,000 Da can rapidly cross the nuclear membrane (Peters, 1984(Peters, , 1986Lang et al, 1986). Increased nuclear fluorescence is also observed with the charged form of ratiometric dyes (Neylon et al, 1990;Christ et al, 1992;Glennon et al, 1992;Neher and Augustine, 1992).…”

Section: Delay and Amplitude Of Nuclear Signalsmentioning

confidence: 75%

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Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion

O'Malley

1994

J. Neurosci.

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In many calcium-imaging studies, the nuclear envelope appears to maintain a gradient of free calcium between the nucleus and cytosol. This issue was examined by loading amphibian sympathetic neurons with the calcium indicator fluo 3 via whole-cell patch clamping. Confocal optical sectioning allowed acquisition of independent calibration curves for the nucleus and cytoplasm. Cells were loaded with free calcium levels ranging from 10 nM to 50 microM, using 10 mM BAPTA to control free calcium. The nuclear fluorescence was usually about 130% brighter than the cytoplasmic fluorescence. Had the increased nuclear fluorescence been due to a calcium gradient, then, as fluo 3 was saturated with calcium in both compartments, the fluorescence gradient should have gradually disappeared. Instead, with free-calcium in the pipette set at 50 microM, about five times the level required to nearly saturate fluo 3, the nuclear/cytoplasmic (N/C) fluorescence ratio was not decreased but instead increased slightly. Perfusion of the patch pipette was used in conjunction with imaging to confirm that cytoplasmic fluo 3 was saturated with calcium. After loading cells with 10 nM free calcium, the patch pipette was perfused with high calcium (10 microM). Again, the N/C fluorescence ratio increased at high calcium. The effectiveness of patch-pipette perfusion in changing cellular free calcium levels was indicated by the degree of fluorescence increase--both nuclear and cytosolic compartments showed a roughly 20-fold increase in fluorescence, that is, most of the dynamic range observed in test droplets. To confirm further that cytoplasmic fluo 3 was saturated, cells were perfused with manganese, which binds with very high affinity to fluo 3. Manganese rapidly entered the cytoplasm and nucleus, causing a large increase in fluorescence, but the N/C fluorescence ratio remained relatively constant. Because free manganese in the pipette was 50,000 times the amount required to saturate fluo 3, the greater nuclear fluorescence probably results from additional fluo 3 in the nucleus rather than from calcium or manganese gradients. To gauge further the permeability of the nuclear envelope, the diffusion of calcium was visualized. Under voltage clamp, calcium channels were opened for periods ranging from 5 to 200 msec. Peak calcium levels were observed within 2 microns of the plasma membrane, and declined as calcium diffused into the cell. The nuclear fluorescence increased more than cytosolic fluorescence, but this apparent “amplification” was eliminated by correcting for autofluorescence. Use of cells cultured on glass coverslips and a high- NA microscope objective allowed a satisfactory correction.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Delay and Amplitude Of Nuclear Signalsmentioning

confidence: 75%

“…Early ratio-imaging experiments, using fura-2, report sustained N/C calcium gradients in smooth muscle (Williams et al, 1985(Williams et al, , 1987Neylon et al, 1990). In these studies, the nucleus rests at a different level of free calcium than the cytosol and appears to be shielded from cytoplasmic calcium transients.…”

mentioning

confidence: 99%

Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion

O'Malley

1994

J. Neurosci.

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“…The 340-380 nm capturing sequence was interrupted by 30-s periods, of which 10 s was used for cell focusing (at 340 nm excitation) and for the remaining 20 s, the shutter was closed, lo avoid bleaching. The MagiCal system has been described in detail by Neylon et al, [17].…”

Section: Fura-2 and Bcecf Loading Of Pt Cellsmentioning

confidence: 99%

Cellular acidification occurs during anoxia in cultured, but not in freshly isolated, rabbit proximal tubular cells

et al. 1995

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In a variety of cells it has been shown that cantly increased cell viability in freshly isolated PT cells acidosis is protective against anoxic injury. We have to 76% ± 5% and 72% ± 4%, respectively (P < 0.05). demonstrated previously that proximal tubule (PT) In contrast, prevention of acidification in cultured PT cells in primary culture were more resistant to anoxia-cells during anoxia did not lead to increased cell death, induced cell injury than were freshly isolated cells. Therefore, the differences in susceptibility to anoxic Therefore, we asked the question of whether a differ-injury between cultured and freshly isolated PT cells ence in cellular acidification during anoxia could ex-cannot be explained by cellular acidification in cultured plain this difference in susceptibility to anoxia. To cells, but must be sought elsewhere, answer this question, intracellular pH (pH;) was meas ured during anoxic incubation of PT cells in culture and those that were freshly isolated. PT cells were incubated in an anoxic chamber at 37°C after load ing with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) or fura-2 acetox y methyl ester (fura-2-AM). pHj and cytosolic free C a2+([Ca2+]i) were measured by digital imaging flu orescence microscopy. During anoxia, pH; in cultured PT cells decreased from 7.3 ±0.1 to 6.8 + 0.1, whereas pH| in freshly isolated cells did not decrease signifi cantly. In addition, the intrinsic buffering capacities (/?, ) in cultured and freshly isolated PT cells were deter-

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“…An epifluorescent 40X magnification oil immersion objective was used. Dynamic video imaging was carried out using the MagiCal hard ware and TARDIS software of Joyce Loebl (Dukesway, Tyne & Wear, UK) as described by Neylon et al (20). The interframe interval between the ratio frames was 6.4 s with a maximal sam pling time of 32 min.…”

Section: Ca2+ Measurementsmentioning

confidence: 99%

Neuropeptide Y inhibits Ca2+ oscillations, cyclic AMP, and secretion in melanotrope cells of Xenopus laevis via a Y1 receptor

et al. 1995

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Spatial dynamics of intracellular calcium in agonist-stimulated vascular smooth muscle cells

Cited by 102 publications

References 6 publications

Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion

Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion

Cellular acidification occurs during anoxia in cultured, but not in freshly isolated, rabbit proximal tubular cells

Neuropeptide Y inhibits Ca2+ oscillations, cyclic AMP, and secretion in melanotrope cells of Xenopus laevis via a Y1 receptor

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