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

Abstract

AbstractIn 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°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 μM; the effect of this anion on [Ca2+]i was mimicked by a decrease of [Ca2+]o to 200 μM in standard [Cl−]o solution. Conversely, titration of [Ca2+]o from 200 μM 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 points[Ca2+]i plays a key role in the metabolic and endocrine functions of white fat adipocytes.In 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.Substitution 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.Growth hormone elevated, [Ca2+]i an effect blocked by inhibitors of voltage-gated Ca2+ channelsIn adipocytes, voltage-gated Ca2+ channel activity appear recalcitrant to changes in Vm, but are however gated by growth hormone.