Intracellular Ca 2+ in Mouse White Fat Adipocytes: Effects of Extracellular Anions,Growth Hormone,and Their Interaction with Ca 2+ Influx

Abstract

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 18 mM 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 18 mM by gluconate substitution decreased [Ca²⁺]_o from 2.6 mM to 200 μM; an effect on [Ca²⁺]_i mimicked by a 152 mM [Cl⁻]_o solution in which [Ca²⁺]_o was 200 μM. Conversely, titration of [Ca²⁺]_o back to 2.6 mM abolished the effect of 134 mM 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 5 mM [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 134 mM [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.