Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the adult central nervous system (CNS), however, it causes excitation in the immature CNS neurons. The shift from GABA-induced depolarization to hyperpolarization in postnatal brain is primarily due to progressive decrease in the expression of the Na-K-2Cl symporter 1 (NKCC1) and increased expression of the K-Cl cotransporter 2 (KCC2). Unlike CNS neurons, both immature and mature neurons in the enteric nervous system (ENS) are depolarized by GABA. Molecular mechanisms by which GABA excites ENS neurons are unclear. It is understood, however, that the excitatory action depends on elevated intraneuronal Cl. We aimed to test a hypothesis that high intracellular Cl in ENS neurons is maintained by activity of the NKCCs. We found that NKCC2 immunoreactivity (IR) was expressed in the ENS of the rat colon on postnatal day 1 (P1). The expression level of NKCC2 continuously increased and reached a steady high level on P14 and maintained at that level in adulthood. NKCC1 IR appeared in ENS on P14 and maintained through adulthood. KCC2 IR was not detectable in the ENS in any of the developmental stages. Both NKCC1 IR and NKCC2 IR were co-expressed with GABA receptors in ENS neurons. Exogenous GABA (1 mmol/L) caused membrane depolarization in the ENS neurons. The reversal potential of GABA-induced depolarization was about -16 mV. Blockade of NKCC by bumetanide (50 μmol/L) or furosemide (300 μmol/L) suppressed the depolarizing responses to GABA. Bumetanide (50 μmol/L) shifted the reversal potential of GABA-induced depolarization in the hyperpolarizing direction. Neither the KCC blocker DIOA (20 μmol/L) nor the Cl/HCO exchanger inhibitor DIDS (200 μmol/L) suppressed GABA-evoked depolarization. The results suggest that ENS neurons continuously express NKCC2 since P1 and NKCC1 since P14, which contribute to the accumulation of Cl in ENS neurons and GABA-evoked depolarization in neonate and adult ENS neurons. These results provide the first direct evidence for the contribution of both NKCC2 and NKCC1 to the GABA-mediated depolarization.
Animals ; Bumetanide ; Neurons ; Rats ; Receptors, GABA-A ; Symporters ; gamma-Aminobutyric Acid

The dried stems of Schisandra henryi var. henryi were extracted with 95% ethanol and the extracts were further subjected to partition, affording the ethyl acetate extracts(EtOAc Extrs.).The EtOAc Extrs.were separated and purified with silica gel and octadecyl-silylated silica gel column chromatography, preparative HPLC and preparative TLC. Thirteen known compounds were obtained and identified by spectral methods including MS and NMR, all of which were elucidated as t-cadinol(1), cadinane-4β,5α,10β-triol(2), cadinane-5α, 10α-diol-2-ene(3), oxyphyllenodiols A(4), 1β, 4β-dihydroxyeudesman-11-ene(5), cyperusol C(6), (7R)-opposit-4(15)-ene-1β,7-diol(7), dysodensiol E(8), epi-guaidiol A(9), aromadendrane-4β,10β-diol(10), tricyclohumuladiol(11), caryolane-1,9β-diol(12), and guaidiol A(13). Compounds 3, 5-10, and 13 were separated from the genus for the first time, while compounds 1-13 were separated from this species for the first time.