Effects of the selective KACh channel blocker NTC-801 on atrial fibrillation in a canine model of atrial tachypacing: comparison with class Ic and III drugs.

The present study examines the effects of NTC-801, a highly selective acetylcholine (ACh) receptor-activated potassium (KACh) channel blocker, on atrial fibrillation (AF) in a canine model with electrical remodeling. An experimental substrate for AF was created in dogs via left atrial (LA) tachypacing (400 bpm, 3-5 weeks). NTC-801, dofetilide, and flecainide were intravenously infused for 15 minutes, and the effects on AF inducibility, atrial effective refractory period (ERP), and atrial conduction velocity were examined. The effect of NTC-801 on AF termination was also evaluated. Atrial ERP was shortened and AF inducibility was increased after LA tachypacing. NTC-801 (0.3-3 µg·kg⁻¹·min⁻¹) prolonged atrial ERP irrespective of stimulation frequency and dose-dependently decreased AF inducibility. Dofetilide (5.3 µg·kg⁻¹·min⁻¹) and flecainide (0.13 mg·kg⁻¹·min⁻¹) did not significantly inhibit AF inducibility and minimally affected atrial ERP. Flecainide decreased atrial conduction velocity, whereas NTC-801 and dofetilide did not. NTC-801 (0.1 mg/kg) converted AF to normal sinus rhythm. In summary, NTC-801 exerted more effective antiarrhythmic effects than dofetilide and flecainide in a canine LA-tachypacing AF model. The antiarrhythmic activity of NTC-801 was probably due to prolonging atrial ERP independently of stimulation frequency. These results suggest that NTC-801 could prevent AF more effectively in the setting of atrial electrical remodeling.

Chymase activities and survival in endotoxin-induced human chymase transgenic mice.

We examined the effects of overexpressed human chymase on survival and activity in lipopolysaccharide (LPS)-treated mice. Human chymase transgenic (Tg) and wild-type C57BL/6 (WT) mice were treated with LPS (0.03, 0.1 and 0.3 mg/day; intraperitoneal) for 2 weeks. Treatment with 0.03 mg LPS did not affect survival in either WT or Tg mice. WT mice were not affected by 0.1 mg/day of LPS, whereas 25% of Tg mice died. Survival of mice treated with 0.3 mg/day of LPS was 87.5% and 0% in WT and Tg, respectively. LPS-induced increases in chymase activity in the heart and skin were significantly greater in Tg than WT mice. These data suggest a possible contribution of human chymase activation to LPS-induced mortality.

Blood glucose level and survival in streptozotocin-treated human chymase transgenic mice.

A growing body of evidence suggests the potential role of chymase in organ injury in diabetes. We investigated blood glucose levels and survival in transgenic mice carrying the human chymase gene (Tg). Intraperitoneal injections of streptozotocin (STZ) (200, 100, 75 and 50 mg/kg in total, i.p.) were given to uninephrectomized Tg mice and wild-type C57BL/6 (BL) mice. Before STZ injection, the Tg mice had significantly lower body weights and slightly higher systolic blood pressure as compared with the BL mice. STZ-treated Tg mice showed significantly higher postprandial blood glucose levels as compared with the STZ-treated BL mice. The survival prevalence of STZ-treated Tg mice was zero, whereas BL mice showed a value of 40% until 42 days. STZ (100, 75 or 50 mg/kg, i.p.)-treated Tg mice also showed a similar pattern as compared with the STZ-treated BL mice. These data suggest that human chymase contributes to blood glucose levels and mortality during the progression of diabetes.

Effects of a highly selective acetylcholine-activated K+ channel blocker on experimental atrial fibrillation.

BACKGROUND: The acetylcholine-activated K(+) current (I(K,ACh)) is a novel candidate for atrial-specific antiarrhythmic therapy. The present study investigates the involvement of I(K,ACh) in atrial fibrillation (AF) using NTC-801, a novel potent and selective I(K,ACh) blocker. METHODS AND RESULTS: The effects of NTC-801, substituted 4-(aralkylamino)-2,2-dimethyl-3,4-dihydro-2H-benzopyran-3-ol, on I(K,ACh) and other cardiac ionic currents (I(Na), I(CaL), I(to), I(Kur), I(Kr), I(Ks), I(Kl), I(KATP), and I(f)) and on atrial and ventricular action potentials were examined in vitro. NTC-801 potently inhibited carbachol-induced I(K,ACh) in guinea pig atrial cells and the GIRK1/4 current in Xenopus oocytes with IC(50) values of 5.7 and 0.70 nmol/L, respectively. NTC-801 selectively inhibited I(K,ACh) >1000-fold over other cardiac ionic currents. NTC-801 (10 to 100 nmol/L) reversed the action potential duration (APD(90)) shortened by carbachol or adenosine in atrial cells, whereas it did not affect APD(90) at 100 nmol/L in ventricular cells. Antiarrhythmic effects of NTC-801 were evaluated in 3 AF models in vivo. NTC-801 significantly prolonged atrial effective refractory period without affecting ventricular effective refractory period under vagal nerve stimulation. NTC-801 dose-dependently converted AF to normal sinus rhythm in both vagal nerve stimulation-induced (0.3 to 3 µg · kg(-1) · min(-1) IV) and aconitine-induced (0.01 to 0.1 mg/kg IV) models. In a rapid atrial pacing model, NTC-801 (3 µg · kg(-1) · min(-1) IV) significantly decreased AF inducibility with a prolonged atrial effective refractory period that was frequency-independent. CONCLUSIONS: A selective I(K,ACh) blockade induced by NTC-801 exerted anti-AF effects mediated by atrial-selective effective refractory period prolongation. These findings suggest that I(K,ACh) may be important in the development and maintenance of AF.

Suppression of CXCR4 expression in mast cells upon IgE-mediated antigen stimulation.

OBJECTIVE: Recent studies have demonstrated that a variety of chemokine receptors are expressed in mast cells. We investigated the changes in mRNA expression of CXCRs in murine IL-3-dependent bone marrow-derived mast cells (BMMCs) to clarify how the CXCR expression is regulated in mast cells. METHODS: Expression of CXCR mRNA was measured by RNase protection assay. Functional expression of CXCRs was confirmed by monitoring intracellular Ca(2+) mobilization. RESULTS: CXCR4 mRNA expression was transiently induced in BMMCs in serum-dependent fashion and was completely suppressed upon IgE-mediated antigen stimulation. In contrast, CXCR5 mRNA expression was induced upon IgE-mediated antigen stimulation. Changes in the intracellular Ca(2+) mobilization induced by CXCL12 strongly indicated the functional expression of CXCR4. The decrease in CXCR4 and the increase in CXCR5 mRNA expression was also observed in BMMCs stimulated with thapsigargin, a phorbol ester, and stem cell factor. CONCLUSION: The mRNA expression of CXCR4 is differentially regulated in BMMCs upon various stimuli including IgE-mediated antigen stimulation.

Interleukin-4-mediated suppression of histamine synthesis in a murine mast cell line, BNu-2cl3.

Since a wide variety of heterogeneity was found in tissue mast cells, recent studies have focused on the process of differentiation and maturation of mast cells. It has been largely accepted that the ability of histamine synthesis is high in the mucosal type mast cells whereas that is low in the tissue-connective type mast cells, although it remains largely unknown how histamine synthesis is regulated during differentiation. Interleukin (IL)-4 is one of the candidate factors that regulate the process of mast cell differentiation. We investigated the effects of IL-4 on histamine synthesis using a murine IL-3-dependent mucosal-type mast cell line, BNu-2cl3. IL-4 drastically suppressed histamine synthesis at the transcriptional levels. Storage of histamine was significantly decreased upon prolonged treatment with IL-4. Down-regulation in expression of histidine decarboxylase by IL-4 was restored by addition of excessive amount of IL-3. Changes in mRNA expression of mouse mast cell proteases (MMCPs) in the cells treated with IL-4 mimicked the differentiation process from mucosal-type to connective tissue-type mast cells; mRNA expression of MMCP2 was decreased, whereas that of MMCP4 and carboxypeptidase A3 were unchanged. These results suggest that IL-4 should play a critical role in suppression of histamine synthesis in mucosal-type mast cells.

Contribution of chymase-dependent angiotensin II formation to the progression of tubulointerstitial fibrosis in obstructed kidneys in hamsters.

Recent studies indicate a role of chymase in the regulation of angiotensin II (AngII) formation in cardiovascular and renal tissues. We investigated a possible contribution of chymase to AngII formation and to renal fibrosis in unilateral ureteral obstruction (UUO). Eight-week-old Syrian hamsters were subjected to UUO and treated with vehicle, the specific chymase inhibitor (CI) 4-[1-(4-methyl-benzo[b]thiophen-3-ylmethyl)-1H-benzimidazol-2-ylsulfanyl]-butyric acid (50 mg/kg, twice a day, p.o.), or the selective AT(1)-receptor blocker olmesartan (10 mg/kg per day, p.o.) for 14 days. UUO-induced renal interstitial fibrosis was associated with increases in renal mRNA levels of alpha-smooth muscle actin (SMA), type I collagen, and transforming growth factor (TGF)-beta. The UUO hamsters showed markedly higher AngII contents and increased AT(1)-receptor mRNA level in the obstructed kidney than sham-operated ones. In contrast, angiotensin-converting enzyme (ACE) protein expression was significantly lower in UUO hamsters. In UUO hamsters, treatment with CI or olmesartan significantly decreased AngII levels in renal tissue and mRNA levels of alpha-SMA, type I collagen, and TGF-beta and ameliorated tubulointerstitial injury. On the other hand, neither CI nor olmesartan changed systolic blood pressure, renal ACE, and AT(1)-receptor protein levels. These data suggest that chymase-dependent intrarenal AngII formation contributes to the pathogenesis of interstitial fibrosis in obstructed kidneys of hamsters.