Characterisation of Ca²⁺ Membrane Permeability of Renal A6 Cells upon Different Osmotic Conditions

Abstract

The nature of the calcium-transporting mechanisms involved in A6 cell calcium homeostasis under iso- and hypo-osmotic conditions was investigated using fura-2 (AM) as a cell calcium indicator. Under steady-state conditions, intracellular calcium (Ca²⁺_i) was increased by Bay K8644 or by gramicidin, an ionophore which depolarises A6 cell membranes. The Ca²⁺_i increase following calcium addition (to calcium-depleted cells) or membrane depolarisation was blocked by nifedipine but not by verapamil or ω-conotoxin, indicating that the membrane calcium permeability may be mediated by voltage-dependent and dihydropyridine-sensitive calcium channels. Ca²⁺_i could also be increased by a hypo-osmotic shock having a linear relationship with the osmolarity change. This osmotically induced Ca²⁺_i increase had an extracellular origin since it was absent when cells were suspended in a calcium-free medium and it was not affected by thapsigargin or TMB-8 application. In addition, it was inhibited by the calcium channel inhibitor, nifedipine. Furthermore, under hypo-osmotic conditions, an additional Ca²⁺_i increase, sensitive to nifedipine, was measured when cells were depolarised by gramicidin or K-gluconate addition. It is proposed that the hypo-osmotically induced cell calcium increase implies the activation of voltage-dependent and nifedipine-sensitive calcium channels, presenting the same pharmacological characteristics as those involved in cell calcium homeostasis under iso-osmotic conditions. The initial Ca²⁺_i increase was transient and stabilised to a value nevertheless higher than the iso-osmotic level; this secondary and incomplete regulatory phase did not occur in the presence of thapsigargin or TMB-8, thus providing evidence of intracellular calcium storage.

Evidence for cadmium mobilization of intracellular calcium through a divalent cation receptor in renal distal epithelial A6 cells

“…Moreover, [DCa 2+ ] Cd was about 64% lower in TG-pretreated cells than in control cells. Since total depletion of internal Ca 2+ stores is very difficult to achieve and, as reported by Brochiero and Ehrenfeld [9], even cell treatment with Ca 2+ -free medium for 10 min does not deplete the internal stores in A6 cells, total Ca 2+ depletion was not expected. Moreover, Cd 2+ may, under these non-physiological conditions of very low or free Ca 2+ i , enter the cells through Ca 2+ channels as the emptying of Ca 2+ i stores stimulates the entry of extracellular Ca 2+ (capacitative calcium entry) [41].…”

Mg2+‐sensitive non‐capacitative basolateral Ca2+ entry secondary to cell swelling in the polarized renal A6 epithelium

“…The polarized nature of the monolayer was demonstrated by (1) electrophysiological characteristics, (2) the absence of changes in [Ca 2+ ] i in response to a reduction of apical osmolality alone, (3) the persistence of Ca 2+ entry in response to a hyposmotic shock in the absence of apical Ca 2+ , (4) the failure of apical Mg 2+ to block Ca 2+ entry during the hyposmotic shock and (5) the differential effect of apical and basolateral ATP on [Ca 2+ ] i elevation under both isosmotic and hyposmotic conditions. Additionally, this study differs from earlier studies on A6 cells (Brochiero & Ehrenfeld, 1997; Yu & Sokabe, 1997; Urbach et al 1999) in that monolayers were grown on permeable supports under conditions that permitted continuous measurement of [Ca 2+ ] i with fura‐2 for periods of up to 60 min.…”

“…In many cell types, cell swelling induces an elevation of [Ca 2+ ] i that is thought to activate mechanisms underlying regulatory volume decrease (Hazama & Okada, 1990; Tinel et al 1994). Osmo‐mechanical stimuli also elevate [Ca 2+ ] i in A6 epithelia (Brochiero & Ehrenfeld, 1997; Yu & Sokabe, 1997; Urbach et al 1999). Hyposmotic stimulation of whole‐cell voltage clamped cells produced [Ca 2+ ] i oscillations involving both Ca 2+ entry and Ca 2+ release from intracellular InsP 3 ‐sensitive stores (Yu & Sokabe, 1997).…”

“…Hyposmotic stimulation of whole‐cell voltage clamped cells produced [Ca 2+ ] i oscillations involving both Ca 2+ entry and Ca 2+ release from intracellular InsP 3 ‐sensitive stores (Yu & Sokabe, 1997). The Ca 2+ entry was shown to occur partly through stretch‐activated channels (Urbach et al 1999) and nifedipine‐sensitive pathways (Brochiero & Ehrenfeld, 1997). Consistent with this observation, another study has demonstrated stretch‐activated Ca 2+ entry in A6 cells by direct application of shear stress (Kawahara & Matsuzaki, 1992).…”

Calcium-induced calcium release participates in cell volume regulation of rabbit TALH cells

“…We can exclude the possibility that membrane stretch is responsible for the activation, as nifedipine completely inhibited the increase of [Ca 2+ ] i . Rather, the channels might belong to the class of dihydropyridine-sensitive channels, which has been found in many other preparations during hypotonic stimulation [3,24].…”