³¹P and ²³Na nuclear magnetic resonance studies of resting and stimulated mast cells

Abstract

Exocytosis induced by crosslinking the type I receptor for Fc, domains present on rat mucosal mast cells (RBL-2H3-line) requires the influx of Ca*+ ions and is markedly influenced by the concentration of monovalent cations (K+, Na+ and protons) in their medium. We investigated the role of these ions in coupling the immunological stimulus to secretion using NMR spectroscopy to monitor simuhanously intracellular pH, ATP and Na+ concentrations and the secretory response of living adherent mast cells. Using this methodology we observed that: (i) ATP concentration and intracellular pH are highly regulated and no changes could be resolved in them upon stimulation and during exocytosis. (ii) In the absence of potassium ions in the cells' medium, a decrease is observed in the intracellular pH and ATP concentration and an increase in the Na+ concentration. (iii) From the influx of extracellular Na+ following inhibition of the Na+, K+-ATPase by ouabain, we estimated the inward Na+ current of resting cells to 5 x 107 ions&-ells). This value does not vary by more than 10% during exocytosis.

86Rb+ ion fluxes in resting and immunologically stimulated mucosal mast cells

“…31P NMR experiments performed on these cells under identical conditions yielded a ratio larger than 10 (within the experimental error no ADP detectable). The ATP concentration did not change upon stimulation of the cells [24], suggesting that the ATP/ADP ratio is maintained at a constant value.…”

“…The cells were perfused with Tyrode buffer prior to each experiment, for at least 1 h and kept at 36°C using a perfusion system developed for NMR experiments on adherent mammalian cells [24,261. The sample was then inserted into the gamma counter and the background counts accumulated every 30 s were registered using an energy window from 800 to 1200 keV required for counting the gamma radiation emitted by x6Rb+ at 1080 keV After allowing sufficient time for background determination, the perfusion buffer was changed to Tyrode that contained additionaly 0.5 pCi/ml x6RbC1 and 0.1 m~ non-radioactive RbCl.…”

“…In our earlier experiments [24] the Na+ influx was measured in media to which Ca2+ ions were added and with inhibition of the Na+/K+-ATPase by ouabain. Under these conditions the Na+ flux was found to be 5(+ 0.1) x lo7 ions/cells.…”

“…This hypothesis is strongly supported also by the findings, that even though Na+ influx takes place in the absence of extracellular Ca2+, no marked increase of intracellular Na+ could be observed upon stimulation of RBL cells when the Na+/K+-ATPase was operating [24, 321. On the other hand, assuming that the Na+/K+-ATPase activity follows a Michaelis Mentenlike dependence on the Na+ concentrations, as was demonstrated for the outer medulla membrane ATPases of the kidney [33], the observed increase in the activity by about 30 % would require at least the same relative increase in the intracellular Na+ concentration.We have earlier studied the intracellular Na+ concentration by NMR [24], but were forced by technical reasons to employ buffers with low sodium concentration. In addition the results show a relatively large experimental error (+ 20) and, therefore, in these experiments the resolution of changes in the intracellular Na+ concentration of about 30 % was almost impossible.…”

“…This methodology was adapted from the one routinely employed for nuclear magnetic resonance (NMR) experiments done on living cells (e.g. [24]). In the course of these experiments we have found that the RBL-2H3 cells can be grown on polymeric beads and that the densely packed cell-covered beads provide high cell concentration (= 3 x 107/ml) that can be maintained alive and studied under continuous perfusion.…”