Cytosolic free calcium in single microdissected rat cortical collecting tubules

Abstract

Cytosolic free Ca²⁺ ([Ca²⁺]_i) was measured in single fragments of rat cortical collecting tubule (CCT) by using fura-2 and a tubule superfusion device. Under basal conditions, i.e. with 1 mM of external Ca²⁺ ([Ca²⁺]_o), the average steady state [Ca²⁺]_i was 179±16 nM (n=44 tubules). Random alterations of [Ca²⁺]_o between 0 mM and 4 mM led to corresponding variations in steady state [Ca²⁺]_i levels, which were linearly correlated with [Ca²⁺]_o (average slope 93±34 nM [Ca²⁺]_i per 1 mM [Ca²⁺]_o for six tubules). In contrast, [Ca²⁺]_i was little affected by decreasing external Na⁺ concentration. Cell membrane depolarization with 100 mM of external K⁺ induced a sustained drop in [Ca²⁺]_i (21% as an average). The data suggest that steady state [Ca²⁺]_i in CCT cells resulted from a non-saturable passive entry of calcium ions across cell membranes balanced with an active extrusion by calcium ATPase (pump and leak mechanism). The passive component cannot be accounted for either by Na⁺/Ca²⁺ exchangers nor by voltage-dependent calcium channels; it is best explained by the presence of voltage-independent calcium channels in cell membranes.