Hasil untuk "physics.acc-ph"

P. Bouton, P. V. Harris, W. Shorthose

H. Marschner, V. Römheld

S. Grinstein, S. Cohen, A. Rothstein

The mechanisms underlying cytoplasmic pH (pHi) regulation in rat thymic lymphocytes were studied using trapped fluorescein derivatives as pHi indicators. Cells that were acid-loaded with nigericin in choline+ media recovered normal pHi upon addition of extracellular Na+ (Nao+). The cytoplasmic alkalinization was accompanied by medium acidification and an increase in cellular Na+ content and was probably mediated by a Nao+/Hi+ antiport. At normal [Na+]i, Nao+/Hi+ exchange was undetectable at pHi greater than or equal to 6.9 but was markedly stimulated by internal acidification. Absolute rates of H+ efflux could be calculated from the Nao+-induced delta pHi using a buffering capacity of 25 mmol X liter-1 X pH-1, measured by titration of intact cells with NH4+. At pHi = 6.3, pHo = 7.2, and [Na+]o = 140 mM, H+ extrusion reached 10 mmol X liter-1 X min-1. Nao+/Hi+ exchange was stimulated by internal Na+ depletion and inhibited by lowering pHo and by addition of amiloride (apparent Ki = 2.5 microM). Inhibition by amiloride was competitive with respect to Nao+. Hi+ could also exchange for Lio+, but not for K+, Rb+, Cs+, or choline+. Nao+/Hi+ countertransport has an apparent 1:1 stoichiometry and is electrically silent. However, a small secondary hyperpolarization follows recovery from acid-loading in Na+ media. This hyperpolarization is amiloride- and ouabain-sensitive and probably reflects activation of the electrogenic Na+-K+ pump. At normal Nai+ values, the Nao+/Hi+ antiport of thymocytes is ideally suited for the regulation of pHi. The system can also restore [Na+]i in Na+-depleted cells. In this instance the exchanger, in combination with the considerable cytoplasmic buffering power, will operate as a [Na+]i- regulatory mechanism.

Karl Werdan, Hans W. Heldt, Mirjana Milovancev

Lawrence D. Mayer, Marcel B. Bally, P. R. Cullis

H. Kontos, A. J. Raper, J. Patterson

T. Arnett, D. Dempster

K. Knauss, T. Wolery

Dongjun Wang, T. Imae

T. Eklund

K. Knauss, T. Wolery

Xi Yu, Zhiqiang Wang, Yugui Jiang et al.

J. Dow

G. R. Bright, G. Fisher, J. Rogowska et al.

Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from populations of cells measured on a cell by cell basis. Therefore, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of cytoplasmic pH. However, the mean cytoplasmic pH can change based on the physiological state of the cells. In addition, there appears to be little, if any, spatial variation in cytoplasmic pH in either quiescent or nonquiescent Swiss 3T3 cells. The pH within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference point for investigating the role of temporal and spatial variations in cytoplasmic pH in a variety of cellular processes including growth control and cell movement.

C. Frelin, P. Vigne, A. Ladoux et al.

M. Hawkins, B. Pope, SutherlandK. Maciver et al.

Gregory E. Morley, S. Taffet, M. Delmar

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.

A. Braghetta, F. Digiano, W. P. Ball

P. Bauerfeind, Rachel M. Garner, B. Dunn et al.

D. L. Fletcher, M. Qiao, D. P. Smith

Three replicate trials were conducted to determine the influence of raw breast meat color and pH on subsequent cooked meat color and pH. In each trial, approximately 50 breast fillets were obtained from a commercial processing plant based on being either normal, lighter than normal, or darker than normal. Color (L* = lightness, a* = redness, and b* = yellowness) of each fillet was determined in triplicate on the underside surface of the fillet (to avoid scalding effects), and the pH was determined on a tissue sample removed from the posterior portion of each fillet. Fillets were then cooked in steam at 98 C for 20 min and cooled to room temperature, and a second sample was removed from the posterior section for cooked meat pH. Cooked meat color was measured on an exposed surface, to avoid cooking-related discoloration. The data were subjected to linear regression analysis to determine the relationship between raw and cooked values. Results indicated a significant linear relationship between raw and cooked values for each color parameter as well as pH. Model R2 values were 0.43, 0.40, 0.64, and 0.78 for L*, a*, b*, and pH, respectively. There were also significant linear relationships between raw meat L* and raw muscle pH (R2 = 0.59) as well as cooked meat L* and raw meat pH (R2 = 0.36). These results indicate that raw breast meat color and pH affect cooked breast meat color and pH but that cooking reduces the degree of color variation. Moreover, cooked meat lightness is more closely associated with raw breast meat pH than with cooked meat pH.