GABA, the main inhibitory neurotransmitter in the adult nervous system, evokes depolarizing membrane responses in immature neurons, which are crucial for the generation of early network activity.
After axotomy, application of muscimol, a GABA(A) receptor agonist, induced an increase in intracellular Ca(2+) ([Ca(2+)](i)) in dorsal motor neurons of the vagus (DMV neurons). Elevation of [Ca(2+)](i) by muscimol was blocked by bicuculline, tetrodotoxin, and Ni(2+). In axotomized DMV neurons measured with gramicidin perforated-patch recordings, reversal potentials of the GABA(A) receptor-mediated response, presumably equal to the equilibrium potential of Cl(-), were more depolarized than that in intact neurons. Thus, GABA(A) receptor-mediated excitation is suggested to be attributable to Cl(-) efflux out of the cell because of increased intracellular Cl(-) concentration ([Cl(-)](i)) in axotomized neurons. Regulation of [Cl(-)](i) in both control and injured neurons was disturbed by furosemide and bumetanide and by manipulating cation balance across the membrane, suggesting that functional alteration of furosemide-sensitive cation-Cl(-) cotransporters is responsible for the increase of [Cl(-)](i) after axotomy. In situ hybridization revealed that neuron-specific K(+)-Cl(-) cotransporter (KCC2) mRNA was significantly reduced in the DMV after axotomy compared with that in control neurons. Similar expression of Na(+), K(+)-Cl(-) cotransporter mRNA was observed between axotomized and control DMV neurons. Thus, axotomy led to disruption of [Cl(-)](i) regulation attributable to a decrease of KCC2 expression, elevation of intracellular Cl(-), and an excitatory response to GABA. A switch of GABA action from inhibitory to excitatory might be a mechanism contributing to excitotoxicity in injured neurons.
GABA is the main inhibitory neurotransmitter in the adult brain. However, GABAergic transmission is depolarizing during early postnatal development, suggesting that changes in the expression of cation-Cl- co-transporters regulating neuronal Cl- homeostasis underlie the ontogeny of GABAergic functions. The developmental changes in the expressions of Cl- co-transporter mRNAs in the neocortex were in opposite directions for NKCC1 (Cl- uptake) and KCC2 (Cl- extrusion). In the newborn, NKCC1 mRNA expression was highest in ventricular zone followed by cortical plate, and then by Layer V/VI, while the reverse was true for KCC2 mRNA. The [Cl-]i levels were in the same rank order as for NKCC1 mRNA. Thus, the ontogeny of Cl- homeostasis in neocortical neurons could be regulated via the differential expression of NKCC1 and KCC2.
Endoplasmic reticulum (ER) stress is profoundly involved in dysfunction of β-cells under high-fat diet and hyperglycemia. Our recent study in mice showed that γ-oryzanol, a unique component of brown rice, acts as a chemical chaperone in the hypothalamus and improves feeding behavior and diet-induced dysmetabolism. However, the entire mechanism whereby γ-oryzanol improves glucose metabolism throughout the body still remains unclear. In this context, we tested whether γ-oryzanol reduces ER stress and improves function and survival of pancreatic β-cells using murine β-cell line MIN6. In MIN6 cells with augmented ER stress by tunicamycin, γ-oryzanol decreased exaggerated expression of ER stress-related genes and phosphorylation of eukaryotic initiation factor-2α, resulting in restoration of glucose-stimulated insulin secretion and prevention of apoptosis. In islets from high-fat diet-fed diabetic mice, oral administration of γ-oryzanol improved glucose-stimulated insulin secretion on following reduction of exaggerated ER stress and apoptosis. Furthermore, we examined the impact of γ-oryzanol on low-dose streptozotocin-induced diabetic mice, where exaggerated ER stress and resultant apoptosis in β-cells were observed. Also in this model, γ-oryzanol attenuated mRNA level of genes involved in ER stress and apoptotic signaling in islets, leading to amelioration of glucose dysmetabolism. Taken together, our findings demonstrate that γ-oryzanol directly ameliorates ER stress-induced β-cell dysfunction and subsequent apoptosis, highlighting usefulness of γ-oryzanol for the treatment of diabetes mellitus.
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