Cell Calcium Levels of Normal and Cystic Fibrosis Nasal Epithelium

Abstract

ABSTRACT. To determine whether epithelial ion transport abnormalities in cystic fibrosis (CF) might reflect abnormal regulation of intracellular Caz+ levels, cytosolic free calcium (Caiz+) was measured using fura-2 or quin2 in suspensions of normal or CF nasal epithelial cells derived from primary cell culture. The basal Ca?+ level measured with fura-2 in CF nasal epithelia was 155 f 9 nM (n = 5), a value not significantly different from normal nasal epithelia (143 f 16 nM, n = 5). Total cell calcium was measured by atomic absorption spectroscopy and no differences were observed between CF (6.3 f 0.5 nmollmg protein; n = 3) and normal (6.2 f 1.2 nmollmg protein; n = 3) nasal epithelial cells. Placing Na+ loaded cells in a low (10 mM) extracellular Na+ solution resulted in a rapid increase in Ca2+ consistent with CaZ+ uptake via a plasmalemmal Na+-Caz+ exchanger. The level of Cai2' achieved by this low Na+ maneuver was not significantly different in CF cells compared to normal cells. Neither isoproterenol (lom5 M) nor forskolin M) had any effect on Ca?+ in normal or CF nasal epithelial cells. Thus, it appears that differences in cell Caiz+, as measured by fluorescent chelators in suspensions of cultured cells, do not account for the abnormalities in basal or isoproterenol stimulated ion transport in CF tissues. (Pediatr Res 24: 79-84,1988) Abbreviations Cai2+, cytolsolic free calcium CF, cystic fibrosis AM, acetoxymethyl ester TCA, trichloroacetic acid PMN, polymorphonuclear cellsThe pathophysiology of CF appears to reflect abnormal epithelial ion transport functions in specific target epithelia (1, 2). Recent evidence has focused on the notion that the abnormalities in epithelial ion transport in the lung reflect dysfunctions in regulatory processes rather than abnormalities in the permeability properties of channel proteins. Evidence for this notion has accrued from two lines of investigation. First, studies of freshly excised nasal epithelia have shown that there is 1) an imbalance between the basal rate of Na+ absorption and the capacity to secrete C1-in CF as compared to normal tissues (3); and 2) the response to P-adrenergic agonists qualitatively differs in the two preparations, i.e., isoproterenol induces C1-secretion in normal

Second‐messenger regulation of sodium transport in mammalian airway epithelia.

“…A23187 has been used as a calcium ionophore in a large number of studies of epithelial ion transport, although measurement of intracellular calcium has been hindered by the fluorescent nature of this compound. However, ionomycin produces very similar effects (Willumsen & Boucher, 1989) and has been shown to directly elevate intracellular calcium in human nasal epithelium (Murphy, Cheng, Yankaskas, Stutts & Boucher, 1988). No stimulation of cyclic AMP levels by A23187 was seen in our tissues.…”

“…However, ionomycin produces very similar effects (Willumsen & Boucher, 1989) and has been shown to directly elevate intracellular calcium in human nasal epithelium (Murphy, Cheng, Yankaskas, Stutts & Boucher, 1988). No stimulation of cyclic AMP levels by A23187 was seen in our tissues.…”

Calcium regulation of ciliary beat frequency in human respiratory epithelium in vitro.

“…The relative effects of recording from spherical cells should be similar with these two cell types, as they are of similar dimensions, but specific differences in intracellular influences on Fura-2 cannot be ruled out. For this reason, although the basal [Ca2+]i measured in our study is of the same order of magnitude as that reported by others (Murphy, Cheng, Yankaskas, Stutts & Boucher, 1988), absolute values obtained could only be taken as best available estimates. However, the relative changes in calcium concentration (expressed as a percentage of baseline) would be unaffected by the lack of absolute calibration standards.…”

Evidence for reduced Cl‐ and increased Na+ permeability in cystic fibrosis human primary cell cultures.

“…This effect of isoprenaline was not observed in CF cell cultures pre-treated with amiloride. The failure of isoprenaline to increase apical membrane Clconductance 4-2 99 R. C. BOUCHER AND OTHERS does not result from an absence of adrenergic receptors, since isoprenaline stimulates cyclic AMP production equally well in CF and normal cultured nasal epithelial cells (Welsh & Liedtke, 1986;Murphy, Yankaskas, Stutts & Boucher, 1988).…”