Modulation of Low-Voltage-Activated Inward Current Permeable to Sodium and Calcium by DARPP-32 Drives Spontaneous Firing of Insect Octopaminergic Neurosecretory Cells

Abstract

Identification of the different intracellular pathways that control phosphorylation/dephosphorylation process of ionic channels represents an exciting alternative approach for studying the ionic mechanisms underlying neuronal pacemaker activity. In the central nervous system of the cockroach Periplaneta americana, octopaminergic neurons, called dorsal unpaired median (DUM; DUM neurons), generate spontaneous repetitive action potentials. Short-term cultured adult DUM neurons isolated from the terminal abdominal ganglion (TAG) of the nerve cord were used to study the regulation of a tetrodotoxin-sensitive low-voltage-activated (LVA) channel permeable to sodium and calcium (Na/Ca), under whole cell voltage- and current-clamp conditions. A bell-shaped curve illustrating the regulation of the amplitude of the maintained current vs. [ATP]i was observed. This suggested the existence of phosphorylation mechanisms. The protein kinase A (PKA) inhibitor, H89 and elevating [cyclic adenosine 3′, 5′ monophosphate, cAMP]i, increased and decreased the current amplitude, respectively. This indicated a regulation of the current via a cAMP/PKA cascade. Furthermore, intracellular application of PP2B inhibitors, cyclosporine A, FK506 and PP1/2A inhibitor, okadaic acid decreased the current amplitude. From these results and because octopamine (OA) regulates DUM neuron electrical activity via an elevation of [cAMP]i, we wanted to know if, like in vertebrate dopaminergic neurons, OA receptor (OAR) stimulation could indirectly affect the current via PKA-mediated phosphorylation of Dopamine- and cAMP-regulated Phosphoprotein-32 (DARPP-32) known to inhibit PP1/2A. Experiments were performed using intracellular application of phospho-DARPP-32 and non-phospho-DARPP-32. Phospho-DARPP-32 strongly reduced the current amplitude whereas non-phospho-DARPP-32 did not affect the current. All together, these results confirm that DARPP-32-mediated inhibition of PP1/2A regulates the maintained sodium/calcium current, which contributes to the development of the pre-depolarizing phase of the DUM neuron pacemaker activity.

Menthol Increases Bendiocarb Efficacy Through Activation of Octopamine Receptors and Protein Kinase A

“…In our experiments, applying of H-89 in the bath eliminated the effect of menthol on the electrical activity of DUM neurons. The stimulation of octopamine receptors in Periplaneta americana causes the activation of adenylyl cyclase, increase of cAMP level, and activation of PKA [29]—factors which reduce the amplitude of mLVA Na + /Ca 2+ current [50]. When the depolarizing current was inhibited, hyperpolarization should occur—this was observed in our experiments.…”

“…We propose the mLVA Na + /Ca 2+ current to be one of the targets for the menthol action. The PKA inhibitor (H-89) has been shown to reverse the effects of octopamine on mLVA Na + /Ca 2+ current [50]. In our experiments, applying of H-89 in the bath eliminated the effect of menthol on the electrical activity of DUM neurons.…”

“…Between LVA Ca 2+ currents, the maintained current permeable to Na + and Ca 2+ (mLVA Na + /Ca 2+ ) has been described in DUM neurons [49]. It plays an essential role in regulating DUM neurons spontaneous discharge frequency [50]. Study of Lapied et al [50] demonstrated that the application of octopamine on isolated DUM neurons decreased the amplitude of mLVA Na + /Ca 2+ via an increase in internal cAMP level and activation of protein kinase A (PKA).…”

“…It plays an essential role in regulating DUM neurons spontaneous discharge frequency [50]. Study of Lapied et al [50] demonstrated that the application of octopamine on isolated DUM neurons decreased the amplitude of mLVA Na + /Ca 2+ via an increase in internal cAMP level and activation of protein kinase A (PKA). PKA activation induces a negative regulatory action on mLVA Na + /Ca 2+ by phosphorylation of DARPP-32 protein.…”