Browsing by UMass Chan Affiliation "Departments of Medicine and Physiology"

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    • Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells
      Ethier, Michael F.; Yamaguchi, Hiroshi; Madison, John M. (2001-06-19)
      In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.

    • Refilling of caffeine-sensitive intracellular calcium stores in bovine airway smooth muscle cells
      Madison, John M.; Ethier, Michael F.; Yamaguchi, Hiroshi (1998-11-14)
      The goal of this study was to assess the mechanisms by which the caffeine-sensitive calcium stores of airway smooth muscle cells are refilled. Bovine trachealis cells were loaded with fura 2-AM (0.5 microM) for imaging of cytosolic calcium concentrations ([Ca2+]i) in the inner cytosol. After a first stimulation (S1) with caffeine, the response to a second stimulation (S2) depended on the presence of extracellular calcium during an intervening 80-s-long refilling phase. The S2-to-S1 ratio (S2/S1) was 0.11 +/- 0.05 (n = 13 cells) during calcium-free refilling but 0.72 +/- 0.04 (n = 36 cells) within 80 s of exposure to extracellular calcium. Maximum mean [Ca2+]i during the 80 s of refilling was not different for calcium-free (116 +/- 19 nM; n = 13 cells) versus extracellular calcium plus nickel (2 mM) (121 +/- 12 nM; n = 21 cells); despite this, significantly greater refilling (S2/S1 0.58 +/- 0.06; n = 24 cells) occurred in the presence of extracellular calcium plus nickel. The protein tyrosine kinase inhibitors genistein (100 microM) and ST-638 (50 microM) significantly decreased refilling over 80 s (S2/S1 0.35 +/- 0.06, n = 14 cells and 0.51 +/- 0.07, n = 14 cells, respectively). Daidzein (100 microM) had no effect on S2/S1. We concluded that [Ca2+]i of the inner cytosol during refilling correlated poorly with S2/S1 values and that, therefore, additional compartments not well detected by fura 2 contribute to refilling. The findings suggest that calcium influx for refilling is segregated from the inner cytosol of the cell, relatively insensitive to nickel, and regulated or modulated by protein tyrosine kinase activity.