Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • BI-7273 Since the Gardos channel Ca sensor have been shown t

    2022-01-20

    Since the Gardos channel Ca2+ sensor have been shown to be constitutively bound calmodulin [16] and the PMCA is activated by association to a calcium–calmodulin complex, it seems probable that the NS309 agonist effect is caused by direct interaction with the Gardos channel, and not by an action on calmodulin per se. This implies, that the activation of the Gardos channels, under the influence of NS309, in the intact cells, are indicators for changes in intracellular free calcium at concentration levels where the Ca2+ pump is almost inactive. A consequence of an activation of the Gardos channel in low K+ Ringers is a concomitant loss of KCl and water, tending to increase the osmotic resistance due to the shrinkage. Contrary to what has been observed at maximum activation of the Gardos channel [17] and the NSVDC channel [18] where the BI-7273 behave as one population, the osmotic resistance curve under the influence of NS309 becomes biphasic, see Fig. 4. The curves seem to be composed of a fraction which increases with time of high resistance cells and a fraction of ‘remaining’, almost unchanged cells (Fig. 5), which points to a fast transit from the normal state to the high resistance state. The fraction of high resistance cells vs. time in the presence of about 4 μM Ca2+ (contamination) and 100 μM NS309 can be fitted to mono exponential functions with time constants of about 70 and 40 min (see Table 1), but with a high resistance fraction at infinite clearly below 1.0. At 100 μM extracellular Ca2+ the high resistance fraction reaches about 0.92 (Fig. 4) and at even higher extracellular Ca2+ the fraction approaches 1.0. It has been shown, that the PMCA capacity for Ca2+ extrusion varies considerably within the cell population [3], from 2–60 mmol/(lcells h) which would cause [Ca2+]cell to vary between 15 and 80 nM under physiological conditions with a Ca2+ influx of 50 μmol/(lcells h) [19]. The NS309 enhanced Gardos channel sensitivity could then cause the cells with the highest Ca content to respond immediately, with subsequent recruitment of cells in the low capacity pumping range. However, under the present experimental conditions, at about 4 μM Ca2+ in the extracellular solution, the mean influx of Ca2+ can be assumed to be appreciably lower, and a graded activation of the Gardos channels in the susceptible fraction should be reflected by a marked broadening of the width of the osmotic resistance distribution for the ‘remaining’ cells, which is not observed (see Fig. 5). An alternative approach to the determination of the osmotic resistance as an estimate of the Gardos channel activation is the measurement of the fraction of cells becoming very dense, in the present experiments cells reaching a density above 1.118, see Fig. 6. It can be calculated, that the cells must loose about 65 mmol KCl/(l original cells) to reach this density. At the normal chloride conductance, which is about 20–25 μS/cm2 [20] the first arrival of cells in the dense state occurs after about 3–4 min. This implies that the flux rate is above 1000 mmol/(l original cells h) of KCl, very close to the flux rate observed at maximum Gardos channel activation. However, the time constant for the exponential increase of the dense fraction is about 50 min, not significantly different from the time constant found for the change in the osmotic resistance at 4 μM extracellular Ca2+. If the chloride conductance is lowered to about 10% of the physiological value by the action of 10 μM NS1652 [21], the first arrival of cells into the dense state is after about 30 min, consistent with the obtainable loss rate at maximum or near maximum Gardos channel activation at a chloride conductance of about 2 μS/cm2, but again with a time constant for the exponential of about 50 min, see Fig. 6. An exponential rise in the high resistance or dense cell fraction is consistent with a random activation of the individual cell. Since Ca2+ is a prerequisite for Gardos channel activation, this could be caused by an opening of a calcium pathway working in an on/off mode, that is channel like. Such a behaviour has been observed previously for sickle cells, run through a series of oxygenation/deoxygenation cycles leading to cells becoming irreversibly sickled, and has been ascribed to the opening of a randomly active cation pathway, Pcat, in deoxygenized cells [22].