1.Na-K-2Cl symporter contributes to γ-aminobutyric acid-evoked excitation in rat enteric neurons.
Sumei LIU ; Lifei ZHENG ; Kayla NEITZEL ; Tuo JI ; Wei REN ; Mei-Hua QU
Acta Physiologica Sinica 2020;72(3):263-273
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
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Bumetanide
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Neurons
;
Rats
;
Receptors, GABA-A
;
Symporters
;
gamma-Aminobutyric Acid
2.Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice.
Jonu PRADHAN ; Dhiraj MASKEY ; Ki Sup PARK ; Myeung Ju KIM ; Seung Cheol AHN
Clinical and Experimental Otorhinolaryngology 2011;4(1):18-23
OBJECTIVES: We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice. METHODS: Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials. RESULTS: Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (Egly) of glycine-induced currents was shifted to a more negative potential by 50 microM bumetanide, a known NKCC1 blocker, and the negatively shifted Egly was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9+/-2.6 mV to -66.0+/-1.5 mV [bumetanide], -66.0+/-1.5 mV to -59.8+/-2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1+/-2.9 mV to -62.7+/-2.6 mV [bumetanide], -62.7+/-2.6 mV to -62.1+/-2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice. CONCLUSION: The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.
Aged
;
Animals
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Brain Stem
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Bumetanide
;
DNA Fingerprinting
;
Furosemide
;
Humans
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Membranes
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Mice
;
Neurons
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Olea
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Symporters
;
Tail
3.Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice.
Jonu PRADHAN ; Dhiraj MASKEY ; Ki Sup PARK ; Myeung Ju KIM ; Seung Cheol AHN
Clinical and Experimental Otorhinolaryngology 2011;4(1):18-23
OBJECTIVES: We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice. METHODS: Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials. RESULTS: Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (Egly) of glycine-induced currents was shifted to a more negative potential by 50 microM bumetanide, a known NKCC1 blocker, and the negatively shifted Egly was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9+/-2.6 mV to -66.0+/-1.5 mV [bumetanide], -66.0+/-1.5 mV to -59.8+/-2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1+/-2.9 mV to -62.7+/-2.6 mV [bumetanide], -62.7+/-2.6 mV to -62.1+/-2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice. CONCLUSION: The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.
Aged
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Animals
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Brain Stem
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Bumetanide
;
DNA Fingerprinting
;
Furosemide
;
Humans
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Membranes
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Mice
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Neurons
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Olea
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Symporters
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Tail
4.Effects of Hydrogen Peroxide on Rabbit Corneal Bioelectric Properties.
Kwang Hyun RYU ; Duk Joon SUH ; Boo Sup OUM
Journal of the Korean Ophthalmological Society 1995;36(8):1395-1406
The rabbit cornea was studied in vitro in modified Ussing chambers to determine the effects of ion transport inhibitors and hydrogen peroxide(H2O2) on ion transport through the cornea by measuring the bioelectric properties. Apical(tear side, T side) addition of furosemide, bumetanide and SITS were ineffective on resting Isc(short circuit current). Apical addition of 1.0mM amiloride(Na+/H+ antiport inhibitor) and NPAA(Cl- channel blocker) markedly reduced the resting Isc, but basolateral(stromal side, S side) addition of amiloride was ineffective. The site of action of these agents was the apical membrane. H2O2, an oxygen free radical, markedly increased the lsc when was added to the T side, but S side addition of the H2O2 was ineffective. To determine the degree of cellular catalase participation in protection against H2O2 induced injury the cornea was pretreated with ATAZ for 30 min prior to H2O2 action. The increase of lsc by H2O2 was markedly potentiated after pretreatment with ATAZ on T side compared to that of S side addition. This result indicates that the corneal endothelial H2O2 may be largely degraded by catalase. When H2O2 was added to the T side, Isc was raised by increased ion transport. All ion transport inhibitors that were used inhibited the H2O2 effect on Isc. Moreover, amiloride and NPAA markedly inhibited induced lsc by H2O2. These results suggest that H2O2 stimulates the corneal epithelial ion transport and that its site of action is apical membrane Na+/H+ antiport system and CI- channel system.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid
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Amiloride
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Bumetanide
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Catalase
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Cornea
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Furosemide
;
Hydrogen Peroxide*
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Hydrogen*
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Ion Transport
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Membranes
;
Oxygen
5.Participation of central GABAA receptors in the trigeminal processing of mechanical allodynia in rats.
Min Ji KIM ; Young Hong PARK ; Kui Ye YANG ; Jin Sook JU ; Yong Chul BAE ; Seong Kyu HAN ; Dong Kuk AHN
The Korean Journal of Physiology and Pharmacology 2017;21(1):65-74
Here we investigated the central processing mechanisms of mechanical allodynia and found a direct excitatory link with low-threshold input to nociceptive neurons. Experiments were performed on male Sprague-Dawley rats weighing 230-280 g. Subcutaneous injection of interleukin 1 beta (IL-1β) (1 ng/10 µL) was used to produce mechanical allodynia and thermal hyperalgesia. Intracisternal administration of bicuculline, a gamma aminobutyric acid A (GABAA) receptor antagonist, produced mechanical allodynia in the orofacial area under normal conditions. However, intracisternal administration of bicuculline (50 ng) produced a paradoxical anti-allodynic effect under inflammatory pain conditions. Pretreatment with resiniferatoxin (RTX), which depletes capsaicin receptor protein in primary afferent fibers, did not alter the paradoxical anti-allodynic effects produced by the intracisternal injection of bicuculline. Intracisternal injection of bumetanide, an Na-K-Cl cotransporter (NKCC 1) inhibitor, reversed the IL-1β-induced mechanical allodynia. In the control group, application of GABA (100 µM) or muscimol (3 µM) led to membrane hyperpolarization in gramicidin perforated current clamp mode. However, in some neurons, application of GABA or muscimol led to membrane depolarization in the IL-1β-treated rats. These results suggest that some large myelinated Aβ fibers gain access to the nociceptive system and elicit pain sensation via GABA(A) receptors under inflammatory pain conditions.
Animals
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Bicuculline
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Bumetanide
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Capsaicin
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gamma-Aminobutyric Acid
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Gramicidin
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Humans
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Hyperalgesia*
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Injections, Subcutaneous
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Interleukin-1beta
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Male
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Membranes
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Muscimol
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Myelin Sheath
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Neurons
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Nociceptors
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Rats*
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Rats, Sprague-Dawley
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Receptors, GABA-A
;
Sensation
6.Effects of Kanagawa Hemolysin on Blood Pressure and Arterial Tone in Rats.
The Korean Journal of Physiology and Pharmacology 2002;6(4):225-
Kanagawa hemolysin (KH), an exotoxin produced from Kanagawa phenomenon-positive Vibrio parahemolyticus, has been shown to possess various biological activities including hemolysis, enterotoxicity, cytotoxicity, and cardiotoxicity. The aim of this study was to investigate the effect of KH on the cardiovascular system and its mechanism, employing in vivo and in vitro experiments of the rat. Intracerebroventricular (icv) administration of 100 mHU KH produced a marked and continuous pressor effect (icv KH-pressor effect), and the icv pressor effect was not repeatable. However, intravenous (iv) injection of the same dose of KH induced a prominent depressor effect (iv KH-depressor effect). The icv KH-pressor effect was inhibited by acid-denaturation, while the iv KH-depressor effect was not. Simultaneous icv administration of the three agents (ouabain, diltiazem, or bumetanide: 10ng/kg each) significantly reduced the pressor effect. The icv KH-pressor effect was inhibited by treatment with iv phentolamine or chlorisondamine, but was not affected by iv candesartan. The iv KH-depressor effect was repeatable and was attenuated by treatment with iv NAME or methylene blue. In vitro experiments using isolated thoracic aorta, 10(-6) M phenylephrine (PE) and 50 mM KCl produced a sustained contraction. In rings contracted with either agents, KH showed relaxant responses in a concentration- dependent fashion and the relaxation (KH-vasorelaxation) was not dependent on the existence of the endothelium. The KH-vasorelaxation in the endothelium-intact rings contracted by PE was abolished by methylene blue treatment. In summary, the present findings suggest that in the icv KH-pressor effect the cation leak-inducing action of KH is implicated, which leads to the increased central sympathetic tone, that the iv KH-depressor effect results from the vasorelaxation via NO-guanylate cyclase system, and that the KH-vasorelaxation is independent of the endothelium and the guanylate cyclase system is involved in it. In conclusion, the mechanism of KH producing the icv pressor effect may not be identical to that of KH producing the iv depressor effect.
Animals
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Aorta
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Aorta, Thoracic
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Blood Pressure*
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Bumetanide
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Cardiovascular System
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Chlorisondamine
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Diltiazem
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Endothelium
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Exotoxins
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Guanylate Cyclase
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Hemolysis
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Ion Transport
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Methylene Blue
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Phentolamine
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Phenylephrine
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Rats*
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Relaxation
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Vasodilation
;
Vibrio
7.cAMP-dependent signalling is involved in adenosine-stimulated Cl- secretion in rabbit colon mucosa.
Sae Ock OH ; Eui Yong KIM ; Jin Sup JUNG ; Jae Skuk WOO ; Yong Keun KIM ; Sang Ho LEE
The Korean Journal of Physiology and Pharmacology 1998;2(4):521-527
An important property of the intestine is the ability to secrete fluid. The intestinal secretion is regulated by a number of substances including vasoactive intestinal peptide (VIP), ATP and different inflammatory mediators. One of the most important secretagogues is adenosine during inflammation. However, the controversy concerning the underlying mechanism of adenosine-stimulated Cl- secretion in intestinal epithelial cells still continues. To investigate the effect of adenosine on Cl- secretion and its underlying mechanism in the rabbit colon mucosa, we measured short circuit current (ISC) under automatic voltage clamp with DVC-1000 in a modified Ussing chamber. Adenosine, when added to the basolateral side of the mucosa, increased ISC in a dose-dependent manner. The adenosine-stimulated ISC response was abolished when Cl- in the bath solution was replaced completely with gluconate. In addition, the ISC response was inhibited by a basolateral Na-K-Cl cotransporter blocker, bumetanide, and by apical Clchannel blockers, dephenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), glibenclamide. Amiloride, an epithelial Na+ channel blocker, and 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS), a Ca2+-activated Cl- channel blocker, had no effect. In the mucosa pre-stimulated with forskolin, adenosine did not show any additive effect, whereas carbachol resulted in a synergistic potentiation of the ISC response. The adenosine response was inhibited by 10 micrometer H-89, an inhibitor of protein kinase A. These results suggest that the adenosine-stimulated ISC response is mediated by basolateral to apical Cl- secretion through a cAMP-dependent Cl- channel. The rank order of potencies of adenosine receptor agonists was 5'-(N-ethylcarboxamino)adenosine(NECA) > N6-(R-phenylisopropyl)adenosine(R-PIA)>2-(p-(2-carbonylethyl)-phenyl-et hylamino)-5'-N-ethylcarboxaminoadenosine(CGS21680). From the above results, it can be concluded that adenosine interacts with the A2b adenosine receptor in the rabbit colon mucosa and a cAMP-dependent signalling mechanism underlies the stimulation of Cl- secretion.
Adenosine
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Adenosine Triphosphate
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Amiloride
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Baths
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Bumetanide
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Carbachol
;
Colforsin
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Colon*
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Cyclic AMP-Dependent Protein Kinases
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Epithelial Cells
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Glyburide
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Inflammation
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Intestinal Secretions
;
Intestines
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Mucous Membrane*
;
Purinergic P1 Receptor Agonists
;
Receptors, Purinergic P1
;
Vasoactive Intestinal Peptide