Intracellular Ca2+ in Mouse White Fat Adipocytes: Effects of Extracellular Anions, Growth Hormone, and Their Interaction with Ca2+ Influx

Background: The membrane potential (Vm) of white fat adipocytes (WFAs) is controlled by passive Cl- permeability. Although electrically unexcitable, WFAs express spontaneously active L-type voltage-gated Ca2+ channels (VGCCs). Consequently, decreases in [Cl-](o), known to depolarize Vm, were explored in their ability to promote VGCC activity and elevate intracellular Ca2+, [Ca2+](i). Materials and Methods: [Ca2+](i) was measured in WFA isolated from epididymal fat of CD-1 mice with epifluorescent Ca2+-imaging techniques. [Ca2+](o) was analyzed by potentiometry. Results: Reduction of [Cl-](o) from 152 to 113, 53 or 18mM by substitution with gluconate or glutamate anions decreased [Ca2+](i) in a graded manner. This effect was due to Ca2+ chelation by the organic anion. Gluconate, glutamate, aspartate, and methylsulphonate had Ca2+ association constants, mean +/- SD (n) of 17 +/- 1.8 (4), 12 +/- 1 (3), 8.2 +/- 4.1 (5) and 3.3 +/- 0.5 L-1 M (5), respectively. Reduction of [Cl-](o) from 152 to 18mM by gluconate substitution decreased [Ca2+](o) from 2.6mM to 200 mu M; an effect on [Ca2+](i) mimicked by a 152mM [Cl-](o) solution in which [Ca2+](o) was 200 mu M. Conversely, titration of [Ca2+](o) back to 2.6mM abolished the effect of 134mM gluconate on [Ca2+](i). Substitution of [Cl-](o) with methylsulphonate did not affect [Ca2+](o) or average [Ca2+](i), although a [Ca2+](i) decrease was seen in WFA with elevated levels. Both basal [Ca2+](i) and the increase observed with 5mM [Ca2+](o) was decreased by verapamil. Twenty nanomolar growth hormone, which activates VGCCs, increased [Ca2+](i), an effect blocked by verapamil, [Ca2+](o) removal, and substitution of 134mM [Cl-](o) with methylsulphonate. Conclusion: The ability of organic anions to chelate Ca2+ as well as for Cl- to modulate L-type VGCC activation complicate experiments that investigate the role of anions in the control of membrane potential and cell function, as is the case for WFA.