Voltage-gated Ca2+ influx in murine white fat adipocytes: stimulation by growth hormone but not membrane depolarization

Abstract

In white fat adipocytes voltage-gated Ca2+ channels are constitutively active. Since the adipocyte membrane potential (Vm) is controlled by Cl- we investigated if changes in [Cl-]o can affect the activity of voltage-gated Ca2+ channels and intracellular calcium, [Ca2+]i. Adipocytes were isolated from epididymal fat of CD-1 mice. [Ca2+]i was imaged with epifluorescent microscopy at 28{degrees}C. Constitutive voltage-gated channel activity was confirmed by the ability of verapamil to decrease [Ca2+]i. Substitution of [Cl-]o to 113, 53 and 18 mM with the membrane impermeant gluconate anion decreased [Ca2+]i from 114{+/-}8.7 to 106{+/-}7.5, 101{+/-}7.1 and 97{+/-}6 nM respectively. Substitution of [Cl-]o with glutamate mimicked the ability of gluconate to decrease [Ca2+]i. To explore if anions affected [Ca2+]i via chelation of external Ca2+, [Ca2+]o was analyzed by potentiometry. Gluconate, glutamate, aspartate and methylsulphonate had Ca2+ association constants of 17{+/-}1.8, 12{+/-}1, 8.2{+/-}4.1 and 3.3{+/-}0.5 L-1 M respectively. Substitution of 134 mM [Cl-]o with gluconate decreased [Ca2+]o from 2.6 mM to 200 M; the effect of this anion on [Ca2+]i was mimicked by a decrease of [Ca2+]o to 200 M in standard [Cl-]o solution. Conversely, titration of [Ca2+]o from 200 M back to 2.6 mM in 134 mM gluconate solutions abolished the effect of this anion on [Ca2+]i. Substitution of [Cl-]o with methylsulphonate to affect Vm did not affect [Ca2+]i. Whereas, growth hormone at 10-20 nM increased [Ca2+]i, an effect blocked by verapamil or absence of [Ca2+]o. In conclusion, growth hormone, but not changes in Vm, can increase voltage-gated Ca2+ channel activity and [Ca2+]i in white fat adipocytes. Key pointsO_LI[Ca2+]i plays a key role in the metabolic and endocrine functions of white fat adipocytes. C_LIO_LIIn adipocytes basal [Ca2+]i is maintained by voltage-gated Ca2+ channels constitutively active at their resting membrane potential, Vm, which is predominantly controlled by Cl- permeability. C_LIO_LISubstitution of [Cl-]o to depolarize Vm with gluconate or glutamate, did not increase but decreased [Ca2+]i. an action due to chelation of extracellular Ca2+. This effect was not seen with methylsulphonate, which did no chelate Ca2+ but did not affect [Ca2+]i. C_LIO_LIGrowth hormone elevated, [Ca2+]i an effect blocked by inhibitors of voltage-gated Ca2+ channels C_LIO_LIIn adipocytes, voltage-gated Ca2+ channel activity appear recalcitrant to changes in Vm, but are however gated by growth hormone. C_LI