Participation of central GABAA receptors in the trigeminal processing of mechanical allodynia in rats

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.

Change in expression of WNK1 in spinal cord of a rat model with bone cancer pain / 中国药理学通报

Aim To investigate the changes in the ex-pression of WNK1 in spinal cord of a rat model with bone cancer pain. Methods Female SD rats, weig-hing 170 ~200 g, were randomly divided into three groups:normal control group (group C, n=3), sham operation group ( group S, n =3 ) and bone cancer pain group ( group BCP, n =24 ) . Group C was not given any treatment, and group S was injected into the bone marrow of left tibia with 5 μl PBS solution while group BCP with 5 μl WALKER 256 mammary gland cancer cell suspension (approximately 1 × 105 cells). Mechanical paw withdrawal threshold ( MWT ) was measured at d1 before inoculation ( baseline) and d3, 6,9,10,11,12 after inoculation. Group S and C were sacrificed at d 12 while group BCP at d 3 ,6 ,9 ,12 after inoculation and spinal cord ( L4~6 ) were removed at different time points for detection of WNK1 mRNA ex-pression by qRT-PCR and WNK1 protein expression by Western blot. Results Compared with group C and S,group BCP’ s MWT started to decrease since d 3 ( P0. 05 ) while the protein expression upregulated since d6 and also showed an in-creasing trend to d 12 ( P<0. 01 ) . Conclusion The expression of WNK1 in spinal cord of a rat model with bone cancer pain increased abnormally, which may be involved in the occurrence and maintenance of a rat model with bone cancer pain.

Ca2+ is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca2+ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca2+ concentration ([Ca2+]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca2+]i increase with nonspecific Ca2+ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca2+]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca2+ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca2+ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca2+ regulation of the WNK-OSR1 pathway in intact cells.

Bumetanide inhibits tumor-cell proliferation by down-regulating NKCC1 expression / 军事医学

Objective To explore the application of bumetanide to inhibition of tumor cell proliferation.Methods In different cell lines, the expression of natrium,kalium, chloride cotransporter 1 ( NKCC1) was detected by Western blotting while the proliferation of different tumor cells was examined by CCK-8 kit.Results The target protein NKCC1 expression in lung cancer cell line ( A549 ) and colorectal cancer cell line ( HCT116 ) was significantly higher than that in chronic myelogenous leukemia cell line (K562), esophageal cancer cell line (Eca109), cervical carcinoma cell line (HeLa), T lymphocytic leukemia cell line (Jurkat) and breast cancer cell line (MCF7).IC50 Values of bumetanide were significantly lower in A549 and HCT116 than in K562, Eca109,HeLa,Jurkat and MCF7.Furthermore, the inhibiory rate and the target protein expression level were positively correlated.Conclusion Bumetanide can inhibit tumor cell proliferation and NKCC1 can serve as a potential target of anticancer drugs.

Decreased Immunoreactivities of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Kanamycin-treated Rats

OBJECTIVES: From our previous study about the weak expressions of potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) in circling mice, we hypothesized that partially damaged cochlea of circling mice might be a cause of the weak expressions of KCC2 or NKCC1. To test this possibility, we reproduced the altered expressions of KCC2 and NKCC1 in the LSO of rats, whose cochleae were partially destroyed with kanamycin. METHODS: Rat pups were treated with kanamycin from postnatal (P)3 to P8 (700 mg/kg, subcutaneous injection, twice a day) and sacrificed for immunohistochemical analysis, scanning electron microscope (SEM) and auditory brain stem response. RESULTS: The SEM study revealed partially missing hair cells in P9 rats treated with kanamycin, and the hearing threshold was elevated to 63.8+/-2.5 dB SPL (4 ears) at P16. Both KCC2 and NKCC1 immunoreactivities were more prominent in control rats on P16. On 9 paired slices, the mean densities of NKCC1 immunoreactivities were 118.0+/-1.0 (control) and 112.2+/-1.2 (kanamycin treated), whereas those of KCC2 were 115.7+/-1.5 (control) and 112.0+/-0.8 (kanamycin treated). CONCLUSION: We concluded that weak expressions of KCC2 and NKCC1 in circling mice were due to partial destruction of cochleae.

The Influence of Sodium salicylate on the Expression of Na+ -K+ -2Cl- / 听力学及言语疾病杂志

Objective To investigate the different expression levels of Na+ - K+ - 2Cl- co- transporter NKCC1 mRNA in the cochlea of rats after sodium salicylate injection and to explore the mechanism underlying the change of outer hair cells, induced by different salicylate administration. Methods Twenty-four normal adult rats were randomly divided into four groups with six rats in each group. Rats in control group,did not recieve sodium sa-licylate injection. The other three groups were acute group,chronic group,and recovered group according to the dif-ferent doses of sodium salicylate. The fluorescence quantitative PCR method was used to detect the expression levels of NKCC1 mRNA in the rat cochleas of the four groups. Results NKCC1 mRNA was expressed in all of the four groups. After sodium salicylate injection, the expressions of NKCC1 mRNA in chronic and recovered group were higher than that in control group(P<0.05). While the expression of NKCC1 mRNA in acute group was lower than that in control group(P(0. 05). Conclusion The expression of NKCC1 mRNA in the normal cochlea indicates that NKCC1 may play an important role in the maintenance of Cl- in the endolymph of the cochlea. The alteration of NKCC1 mRNA expression caused by sodium salicylate injection may lead to the change of the outer hair cell electro-motility.

Differential expression of Na+-K+-Cl- cotransporter 1 and K+-Cl- cotransporter 2 in the somata and dendrites of adult rat neocortical neurons[1] / 神经解剖学杂志

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the adult brain. However, electrophysiological findings indicate that GABA exerts excitation in dendrites of mature eorlieal neurons. Little is known about morphological basis of GABA-medi-ated excitation in dendrites of mature cortical neurons. The effect of activated GABAA receptors is mainly determined by intraceUular chloride ion, whose active influx is mainly mediated by Na+-K +-Cl- cotransporter isoform 1 (NKCC1) and exclusion is mainly executed by K+-Cl- cotransporter isoform 2 ( KCC2 ). In the present study, by using immunofluorescent double staining and fluorescent density analysis, the expression and distribution of NKCCI- and KCC2-immunoreactivities in the dendrite and soma of adult rat neocortical neurons were detected in vivo and in vitro. The present results showed that both cytoplasm and membrane of neuronal soma and dendrite expressed NKCC1, while KCC2 only expressed in membrane of soma and dendrite. The results also indicated that the dendrites rather than the somata of neurons expressed more NKCC1 in adult rat neocortex, while the level of KCC2 expression in the dendrite membrane was similar to that in the membrane of somata. The similar expression pattern of NKCC1 and KCC2 in the dendrites and the somata was also observed in neocortical neurons cultured for 20 days in vitro. The present results suggest that the more NKCC1 expreasion in dendrites may contribute to GABA-mediated excitation in neuronal dendrites of adult neocortex neurons.

Contributory Roles of Distal Renal Sodium Transporters in NSAID-induced Sodium Retention / 대한신장학회잡지

BACKGROUND: Sodium retention occurs in some patients taking NSAIDs (nonsteroidal anti-inflammatory drugs). Although the renal effects of NSAIDs are predominantly mediated through the inhibition of prostaglandins synthesized by cyclooxygenase-2 (COX-2), the mechanisms of sodium retention are not clear at the sodium transporter levels in the kidney. Previous studies have shown that compensatory upregulation of COX-2 is induced in renal medulla by high salt intake and that NSAID-induced sodium retention may be transitory. METHODS: To investigate whether renal sodium transporter abundances are altered by NSAID administration and whether renal sodium transporter abundances are affected by high salt intake or chronic NSAID administration, we performed an acute study treated with a single injection of diclofenac and another chronic study treated with 7 days' administration of DFU, a selective COX-2 inhibitor, using semiquantitative immunobotting from rat kidneys. Male Sprague-Dawley rats were divided into three groups in each study: controls, NSAID treatment, and high-salt intake plus NSAID treatment. The control diet contained sodium 1 mmol/200 g BW/day, and the high-salt diet 10 mmol/200 g BW/day. RESULTS: The acute study using diclofenac (100 mg/kg BW) increased the abundances of NKCC2 (by 73%) and ENaC-alpha (by 60%) in cortex and of NKCC2 (by 165%) and ENaC-alpha (by 91%) in outer medulla, in association with a significant decrease in urinary sodium excretion. The increased ENaC-alpha abundance was reversed by addition of high salt intake in both cortex and outer medulla. The chronic study using DFU (40 mg/kg/d for 7 days) showed no significant changes in distal renal sodium transporters except a decreased abundance of Na-K- ATPase alpha1-subunit (by 24%) in outer medulla. The addition of high salt intake decreased the abundances of ENaC-alpha (by 35%) and ENaC-beta (by 47 %) in outer medulla. CONCLUSION: The abundances of thick ascending limb NKCC2 and collecting duct ENaC are altered in response to NSAID administration. It is suggested that NKCC2 & ENaC are contributory to NSAID- induced sodium retention and also have a compensatory role in high salt intake and chronic NSAID administration.

Chronic Furosemide Administration Alters Abundance of Sodium Transporters in Rat Kidney / 대한신장학회잡지

BACKGROUND: Furosemide inhibit NaCl absorption in the thick ascending limb and produce an increase in distal delivery of Na+. We carried out semiquantitative immunoblotting and immunohistochemistry of rat kidneys to investigate whether chronic furosemide infusion is associated with compensatory increases in the abundance of Na+ transporters in distal nephron. METHODS: Osmotic minipumps were implanted into Sprague-Dawley rats to deliver 12 mg/day of furosemide(n=6) with simultaneous administration of 0.8% NaCl and 0.1% KCl in drinking water for 7 days. RESULTS: Compared with vehicle infused controls, urine volume and urine sodium amount were increased. However, there were no differences in body weight, serum aldosterone, and creatinine clearance. The abundance of Na+-K+-2Cl- cotransporter after furosemide infusion was increased in cortex (151+/-10 vs. 100+/-10%, p< 0.05) and outer medulla (122+/-5 vs. 100+/-3%, p< 0.01). In furosemide infusion group, the abundance of all three subunits of epithelial sodium channel (ENaC) was increased both in cortex (alpha: 187+/-25 vs. 100+/-17%, p< 0.05; beta: 155+/-8 vs. 100+/-15%, p< 0.05; gamma: 168+/-16 vs. 100+/-9%, p< 0.05) and outer medulla (alpha: 171+/-27 vs. 100+/-17%, p< 0.05; beta: 986+/-91 vs. 100+/-33%, p< 0.01; gamma: 242+/-24 vs. 100+/-22%, p< 0.01). Consistent with these results, ENaC beta-subuint immunohistochemistry showed a remarkable increase in immunoreactivity in the principal cells of collecting ducts with furosemide treatment. CONCLUSION: These increases in the abundance of ENaC protein may account for the generation of diuretic tolerance.

Application and Immune Effect of Nutrison-5Fu on Perioperative Patients with Progressive Gastrointestinal Cancer / 中国医师杂志

Objectives To investigate and evaluate the effect of Nutrison-5Fu on immune function of perioperative patients with progressive gastrointestinal cancer,and simultaneously we also recommended the methods of application of Nutrison-5Fu.Methods Seventy eight cases were equally divided at random into three groups,26 cases in each group.⑴Nutrison-5Fu group:the patients were treated with Nutrison-5Fu.⑵Chemotherapy group:the patients only received 5-Fu.⑶Control group:there were not any chemotherapy and immunetherapy during the perioperation.The values of CD 3,CD 4,CD 8 and NKCC were determined in all cases.Results Values of CD 3,CD 4,CD/CD 8 ratio and NKCC in most patients were distinctly low,but the condition rapid improved by giving them Nutrison-5Fu.On the contrary,the values declined further after chemotherapy alone.Conclusion The treatment of Nutrison-5Fu not only can repress the development of the tumor,but also can improve the dystrophic status and immune function of the patients with cancer.

Deficiency in Na-K-2Cl co-transporter impaired hearing and balance in mice / 中国病理生理杂志

AIM:We generated transgenic mice of NKCC1-/-(homozygous mutant),NKCC1+/-(heterozygous)and NKCC1+/+(wild-type)that have a targeted disruption in the NKCC1 gene to investigate the role of Na-K-2Cl(NKCC1)channel in auditory function of the inner ear.METHODS:Hearing threshold and endocochlear potential(EP)were measured in the NKCC1-/-,NKCC1+/-and NKCC1+/+ mice by auditory brainstem response(ABR)and EP recordings,respectively.The inner ears of the mice were removed and examined morphologically with the light microscope.RESULTS:The auditory function of NKCC1+/+ mice was normal,the mean value for ABR thresholds in response to click sound was [(23.13?3.78)dB,SPL],EP was(98?16)mV.The mean value for ABR thresholds to click sound was elevated in NKCC1+/-mice [(38.49?12.29)dB,SPL],relative to that significantly increased in NKCC1+/+ mice(P

The Influence of Sodium Salicylate on the Expression of Na~+-K~+-2Cl~- / 听力学及言语疾病杂志

Objective To investigate the different expression levels of Na+-K+-2Cl-co-transporter NKCC1 mRNA in the cochlea of rats after sodium salicylate injection and to explore the mechanism underlying the change of outer hair cells,induced by different salicylate administration.Methods Twenty-four normal adult rats were randomly divided into four groups with six rats in each group. Rats in control group,did not recieve sodium salicylate injection. The other three groups were acute group,chronic group,and recovered group according to the different doses of sodium salicylate.The fluorescence quantitative PCR method was used to detect the expression levels of NKCC1 mRNA in the rat cochleas of the four groups.Results NKCC1 mRNA was expressed in all of the four groups.After sodium salicylate injection,the expressions of NKCC1 mRNA in chronic and recovered group were higher than that in control group(P