Effects of acetazolamide on transient K+ currents and action potentials in nodose ganglion neurons of adult rats.

The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K(+) current (I(A) ) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4-AP attenuated or blocked the CO(2) -induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch-clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT-PCR of mRNA and the expression of voltage-gated K(+) (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the I(A) by approximately 27% and the additional application of 4-AP (1 mM) further inhibited I(A) by 48%. The application of 0.1 µM α-dendrotoxin (α-DTX), a slow inactivating transient K(+) current (I(D) ) blocker, inhibited the baseline I(A) by approximately 27%, and the additional application of 1 mM AZ further decreased the I(A) by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage-gated K(+) channel proteins were present, and most (80-90%) of the four Kv channels immunoreactive neurons showed the co-expression of carbonic anhydrase-II (CA-II) immunoreactivity. These results indicate that the application of AZ causes the reduction in I(A) via the inhibition of four voltage-gated K(+) channel (Kv) proteins without affecting I(D).

Capture of type 1 diabetes-susceptible HLA DR-DQ haplotypes in Japanese subjects using a tag single nucleotide polymorphism.

OBJECTIVE: To identify type 1 diabetes-susceptible HLA DR-DQ haplotypes using tag single nucleotide polymorphisms (SNPs) and to estimate the disease risk using these tag SNPs. RESEARCH DESIGN AND METHODS: Five tag SNPs were typed in a total of 211 Japanese subjects including 201 patients with type 1 diabetes who had already been typed for HLA-DRB1, -DQA1, and -DQB1 alleles and 300 control subjects. RESULTS: Tag SNP rs2395185 captured haplotypes involving all DR4 specificities and DR9 specificity with a sensitivity of 98.5% and specificity of 94.9%. Using the T allele of rs2395185, we obtained an odds ratio (95% CI) of 2.87 (2.21-3.74) for type 1 diabetes. In addition, rs3129888 captured haplotypes involving HLA-DRB1*0802 with a sensitivity of 92.3% and specificity of 98.9%. CONCLUSIONS: Typing of two tag SNPs (rs2395185 and rs3129888) may be useful for the screening of Japanese subjects at genetic risk of type 1 diabetes.

Effect of 8-bromo-cAMP on the tetrodotoxin-resistant sodium (Nav 1.8) current in small-diameter nodose ganglion neurons.

We examined whether 8-bromo-cAMP (8-Br-cAMP)-induced modification of tetrodotoxin-resistant (TTX-R) sodium current in neonatal rat nodose ganglion neurons is mediated by the activation of protein kinase A (PKA) and/or protein kinase C (PKC). In 8-Br-cAMP applications ranging from 0.001 to 1.0mM, 8-Br-cAMP at 0.1mM showed a maximal increase in the peak TTX-R Na(+) (Nav1.8) current and produced a hyperpolarizing shift in the conductance-voltage (G-V) curve. The PKC inhibitor bisindolylmaleimide Ro-31-8425 (Ro-31-8425, 0.5microM) decreased the peak Nav 1.8 current. The Ro-31-8425-induced modulation of the G(V)(1/2) baseline (a percent change in G at baseline V1/2) was not affected by additional 8-Br-cAMP application (0.1mM). The maximal increase in Nav 1.8 currents was seen at 0.1microM after the application of a PKC activator, phorbol 12-myristate 13-acetate (PMA) and forskolin. The PMA-induced increase in Nav 1.8 currents was not significantly affected by additional 0.1mM 8-Br-cAMP application. Intracellular application of a PKA inhibitor, protein kinase inhibitor (PKI, 0.01mM), inhibited the baseline Nav 1.8 current, significantly attenuated the 8-Br-cAMP-and PMA-induced increase in the peak Nav 1.8 current, and caused a significant increase in the slope factor of the inactivation curve. The PKI application at a higher concentration (0.5mM) greatly inhibited the PMA (0.1microM)-induced increase in the peak Nav 1.8 current amplitude and further enhanced the Ro-31-8425-induced decrease in the current. These results suggest that the 8-Br-cAMP-induced increase in Nav 1.8 currents may be mediated by activation of both PKA and PKC.

Association between intronic SNP in urate-anion exchanger gene, SLC22A12, and serum uric acid levels in Japanese.

Serum uric acid levels are maintained by urate synthesis and excretion. URAT1 (coded by SLC22CA12) was recently proposed to be the major absorptive urate transporter protein in the kidney regulating blood urate levels. Because genetic background is known to affect serum urate levels, we hypothesized that genetic variations in SLC22A12 may predispose humans to hyperuricemia and gout. We investigated rs893006 polymorphism (GG, GT and TT) in SLC22A12 in a total of 326 Japanese subjects. Differences in clinical characteristics among the genotype groups were tested by the analysis of variance (ANOVA). In male subjects, mean serum uric acid levels were significantly different among the three genotypes. Levels in the GG genotype subjects were the highest, followed by those with the GT and TT genotypes. However, no differences between the groups were seen in the distributions of creatinine, Fasting plasma glucose (FPG), HbA(1c), total cholesterol, triglyceride, HDL cholesterol levels or BMI. A single nucleotide polymorphism (SNP) in the urate transporter gene SLC22CA12 was found to be associated with elevated serum uric acid levels among Japanese subjects. This SNP may be an independent genetic marker for predicting hyperuricemia.

Temporomandibular joint inflammation potentiates the excitability of trigeminal root ganglion neurons innervating the facial skin in rats.

The aim of this study was to test the hypothesis that temporomandibular joint (TMJ) inflammation alters the excitability of trigeminal root ganglion (TRG) neurons innervating the facial skin, by using behavioral, electrophysiological, molecular, and immunohistochemical approaches. Complete Freund's adjuvant (CFA) was injected into the rat TMJ to produce inflammation. The threshold for escape from mechanical stimulation applied to the orofacial area in TMJ-inflamed rats was significantly lower than that in naïve rats. The TRG neurons innervating the inflamed TMJ were labeled by 2% Fluorogold (FG) injection into the TMJ. The number of FG-labeled substance P (SP)-immunoreactive neurons in the inflamed rats was significantly increased compared with that in the naïve rats. On the other hand, medium- and large-diameter TRG neurons (>30 microm) innervating the facial skin were labeled by FG injection into the facial skin. In the FG-labeled cutaneous TRG neurons, the occurrence of SP (100 nM) induced membrane depolarization in inflamed rats (medium: 73.3%, large : 85.7%) was larger than that in the naïve rats (medium: 29.4%, large : 0%). In addition, SP application significantly increased the firing rate evoked by depolarizing pulses in the neurons of inflamed rats compared with those of naïve rats. Quantitative single-cell RT-PCR analysis showed the increased expression of mRNA for the NK1 receptor in FG-labeled TRG neurons in inflamed rats compared with that in naive rats. The numbers of SP and NK1 receptors/neurofilament 200 positive immunoreactive TRG neurons innervating the facial skin (FG-labeled) in the inflamed rats were significantly increased compared with those seen in naïve rats. These results suggest that TMJ inflammation can alter the excitability of medium- and large-diameter TRG neurons innervating the facial skin and that an increase in SP/NK1 receptors in their soma may contribute to the mechanism underlying the trigeminal inflammatory allodynia in the TMJ disorder.

Association of the SNP-19 genotype 22 in the calpain-10 gene with elevated body mass index and hemoglobin A1c levels in Japanese.

BACKGROUND: An association of variations in the calpain-10 gene (CAPN10) with type 2 diabetes was originally reported in Mexican Americans. However, some studies in other racial groups were contradictory. METHODS: We studied the influence of genotypes and haplotype combinations (haplogenotypes) of CAPN10 on the metabolic traits in 286 Japanese subjects who visited a General Health Check-up Center. RESULTS: As for single nucleotide polymorphism (SNP)-19, subjects with genotype 22 had higher body mass index (BMI) and hemoglobin A(1c) (HbA(1c)) levels than those with genotypes 11 and 12 (p=0.003 and p=0.024, respectively). In SNP-63, subjects with genotype 11 had higher BMI and HbA(1c) levels than those with genotypes 12 and 22 (p=0.003 and p=0.045, respectively). Subjects who had genotype 22 at SNP-19 always had genotype 11 at SNP-63. Subjects with haplogenotype 121/121 had higher BMI levels (p=0.021) and tended to have higher HbA(1c) levels (p=0.08) than those with other haplogenotypes. All things considered, SNP-19 genotype 22, which was composed of haplogenotype 121/121 and 121/221, had highest significance level of association with elevated HbA(1c) levels. SNP-43 did not affect metabolic traits in our subjects. Subjects with haplogenotype 112/121 rather showed lower BMI and HbA(1c) levels (p=0.016 and p=0.008) than those with all other haplogenotypes. CONCLUSIONS: These results indicate the contribution of SNP-19 in CAPN10 to mild obesity and glucose intolerance in Japanese.