Although aerobic exercise has been shown to lower blood pressure (BP) in human beings, its additive BP-reducing effect on antihypertensive drug therapy seems to have been investigated in only laboratory animals.This study investigated the effects of aerobic dance combined with antihypertensive drugs on BP and number of antihypertensive drugs in individuals with hypertension.This open label randomised-controlled trial involved new-diagnosed male and female individuals with mild-to-moderate essential hypertension after at least four weeks of treatment. They were randomly assigned to drug therapy (Normoretic: Hydrochlorothiazide + amiloride hydrochloride, and Amlodipine) (control: n=33) and aerobic dance combined with drug therapy (exercise: n=30) groups. Intervention in each group lasted 12 weeks. BP was measured at baseline and during and pos-intervention. Number of antihypertensive drugs was recorded post-intervention.There were significant reductions in SBP at some periods of the intervention in the exercise group (p=0.000 to 0.002) and control group (p=0.001 to 0.002), and significant difference in DBP at some periods of the intervention in exercise group (p=0.000 to 0.003) and control group (p=0.000 to 0.001). SBP (p=0.066) and DBP (p=0.100) did not differ between the two groups post-12-week intervention. The BP control rates were similar between the exercises (56.7%) and control (35.5%) groups (p=0.075). Similarly, between-group difference in the number of drugs was not significant (p=0.511).This preliminary report demonstrates the tendency of aerobic dance to enhance BP control in individuals on two antihypertensive drugs without BP control.

Activation of the nerve growth factor (NGF) receptor trkA and tissue acidosis are critically linked to inflammation-associated nociceptor sensitization. This study explored how increased acidity is linked to sensory neuron sensitization to NGF. Adult Wistar rat primary sensory neurons grown at physiological pH 7.4, then either kept at pH 7.4 or challenged for 30 min in pH 6.5 medium, provided a model of acidosis. Nonpermeabilizing trkA immunofluorescence revealed a significant increase in trkA mobilization to the plasma membrane from intracellular stores in response to proton challenge. This was confirmed using a surface protein biotinylation assay and Brefeldin A disruption of the rough endoplasmic reticulum-Golgi-trans-Golgi network. Mobilization of trkA to the membrane at pH 6.5 was abolished in neurons treated with the acid-sensitive ion channel blocker, amiloride. While elevated levels of NGF-independent trkA phosphorylation occurred at pH 6.5 alone, the level of activation was significantly increased in response to NGF challenge. Exposure of sensory neurons to pH 6.5 medium also resulted in strong calcium (Ca(2+)) transients that were reversible upon reintroduction to physiological pH. The pH 6.5-induced mobilization of trkA to the membrane was Ca(2+) dependent, as BAPTA-AM Ca(2+) chelation abrogated the response. Interestingly, KCl-induced depolarization was sufficient to induce mobilization of trkA to the cell surface at pH 7.4, but did not augment the response to pH 6.5. In conclusion, increased mobilization of trkA to neuronal membranes in response to either acidosis or neuronal depolarization provides two novel mechanisms by which sensory neurons can rapidly sensitize to NGF and has important implications for inflammatory pain states.

BACKGROUND:

Acid-sensing ion channels (ASICs) are esophageal nociceptors that are candidates to mediate heartburn in non-erosive reflux disease (NERD). Amiloride, a diuretic, is known to inhibit ASICs. For this reason, we sought a role for ASICs in mediating heartburn by determining whether amiloride could block heartburn in NERD induced by esophageal acid perfusion.

METHODS:

In a randomized double-blind crossover study, we perfused the esophagus with amiloride or (saline) placebo prior to eliciting acid-induced heartburn in patients with a history of proton pump inhibitor-responsive NERD. Those with NERD and positive modified Bernstein test were randomized to perfusion with amiloride, 1 mmol/l, or placebo for 5 min, followed by repeat acid-perfusion. Heartburn severity and time to onset was measured and the process repeated following crossover to the alternative agent.

RESULTS:

14 subjects completed the study. Amiloride did not reduce the frequency (100 vs. 100 %) or severity of acid-induced heartburn (Mean 2.50 ± SEM 0.33 vs. 2.64 ± 0.45), respectively. There was a trend towards longer time to onset of heartburn for amiloride versus placebo (Mean 2.93 ± SEM 0.3 vs. 2.36 ± 0.29 min, respectively), though these differences did not reach statistical significance (p > 0.05).

CONCLUSIONS:

Amiloride had no significant effect on acid-induced heartburn frequency or severity in NERD, although there was a trend towards prolonged time to onset of symptoms.

The epithelial sodium channel (ENaC) has four subunits, namely α (alpha), β (beta), γ (gamma) and δ (delta). The functional ENaC is formed by the combination of either αβγ or δβγ subunits. The aim of the present study is to determine the combination of ENaC subunits predominant on the apical side of the frog skin, and the effect of ADH on sodium transport though these two ENaCs subunit combinations. The ventral abdominal skin of the frog, Rana hexadactyla was mounted in an Ussing-type chamber. The voltage-clamp method was performed to measure the ionic transport across the frog skin with normal Ringer solution (NR) on both sides. Evans blue (300 µM) and amiloride (100 µM) were added to the NR on the apical side and ADH (40 nM) was added on the serosal side. Statistical significance was analyzed by Student's paired t-test and repeated-measures ANOVA, P < 0.05 was considered significant. This study suggests that the ENaC of the frog skin consist of both αβγ and δβγ subunit combinations on the apical side. Though both types of subunit combination are present, the αβγ type was found to be more common than δβγ. ADH increases the sodium transport across the frog skin. The effect of ADH on sodium transport is achieved through the combination of δ-subunits, not through the combination of a-subunits in the skin of Pana hexadactyla.

Amiloride is a potassium-sparing diuretic that has been used as an anti-kaliuretic for the chronic management of hypertension and heart failure. Several studies have identified a potential anti-cancer role for amiloride, however the mechanisms underlying its anti-tumor effects remain to be fully delineated. Our group previously demonstrated that amiloride triggers caspase-independent cytotoxic cell death in human glioblastoma cell lines but not in primary astrocytes. To delineate the cellular mechanisms underlying amiloride's anti-cancer cytotoxicity, cell permeant and cell impermeant derivatives of amiloride were synthesized that exhibit markedly different potencies in cancer cell death assays. Here we compare the cytotoxicities of 5-benzylglycinyl amiloride (UCD38B) and its free acid 5-glycinyl amiloride (UCD74A) toward human breast cancer cells. UCD74A exhibits poor cell permeability and has very little cytotoxic activity, while UCD38B is cell permeant and induces the caspase-independent death of proliferating and non-proliferating breast cancer cells. UCD38B treatment of human breast cancer cells promotes autophagy reflected in LC3 conversion, and induces the dramatic swelling of the endoplasmic reticulum, however these events do not appear to be the cause of cell death. Surprisingly, UCD38B but not UCD74A induces efficient AIF translocation from the mitochondria to the nucleus, and AIF function is necessary for the efficient induction of cancer cell death. Our observations indicate that UCD38B induces programmed necrosis through AIF translocation, and suggest that its cytosolic accessibility may facilitate drug action.

Alveolar fluid clearance driven by active epithelial Na(+) and secondary Cl(-) absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na(+) channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl(-) secretion and alveolar Cl(-) influx were quantified by radionuclide tracing and alveolar Cl(-) imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl(-) secretion and alveolar Cl(-) influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na(+)-K(+)-Cl(-) cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR(-/-) mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl(-) secretion that were again CFTR-, NKCC-, and Na(+)-K(+)-ATPase-dependent. Our findings show a reversal of transepithelial Cl(-) and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl(-) and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema.

TRPM5 is involved in sensing sweet, bitter, umami and fat taste stimuli, complex tasting divalent salts and temperature-induced changes in sweet taste. To investigate if the amiloride- and benzamil (Bz)-insensitive NaCl chorda tympani (CT) taste nerve response is also regulated in part by TRPM5, CT responses to 100 mM NaCl+5 μM Bz (NaCl+Bz) were monitored in Sprague-Dawley rats, wildtype (WT) mice, and TRPV1 and TRPM5 knockout (KO) mice, in the presence of resiniferatoxin (RTX), a TRPV1 agonist. In rats, NaCl+Bz+RTX CT responses were also monitored in the presence of triphenylphosphine oxide (TPPO), a specific TRPM5 blocker and capsazepine (CZP) and N-(3-methoxyphenyl)-4-chlorocinnamid (SB-366791), specific TRPV1 blockers. In rats and WT mice, RTX produced biphasic effects on the NaCl+Bz CT response, enhancing the response between 0.5 and 1 μM and inhibiting it above 1 μM. In rats and WT mice, NaCl+Bz+SB-366791 CT response and in TRPV1 KO mice the NaCl+Bz CT response was inhibited to baseline level and was RTX insensitive. In rats, blocking TRPV1 by CZP or TRPM5 by TPPO inhibited the tonic NaCl+Bz CT response and shifted the relationship between RTX concentration and the magnitude of the tonic CT response to higher RTX concentrations. TRPM5 KO mice elicited no constitutive NaCl+Bz tonic CT response. The RTX concentration versus the magnitude of the tonic NaCl+Bz CT response relation was significantly attenuated and shifted to higher RTX concentrations. The results suggest that altering TRPM5 activity pharmacologically or genetically modulates the Bz-insensitive NaCl CT response and its modulation by TRPV1 agonists.

The epithelial sodium channel (ENaC)(1) plays an important role in controlling Na(+) homeostasis, extracellular fluid volume and blood pressure. Copper Metabolism Murr1 Domain-containing protein 1 (COMMD1) interacts with ENaC and down-regulates ENaC. COMMD1 belongs to the COMMD family consisting of COMMD1-10, and all COMMD family members share a C-terminal COMM domain. Here, we report that COMMD2-10 also interact with ENaC, and COMMD3 and COMMD9 were selected for further study. Amiloride-sensitive current in mammalian epithelia expressing ENaC was significantly reduced by COMMD3 or COMMD9, and ENaC expression at the cell surface was significantly decreased in the presence of COMMD3 or COMMD9. COMMD3 and 9 retained their ability to reduce current when COMMD1 was knocked down. COMMD3 and COMMD9 were widely expressed in kidney and were colocalized with ENaC in renal collecting duct cells. These data suggest that COMMD3 and COMMD9 may be endogenous regulators of ENaC to regulate Na+ transport through altering ENaC cell surface expression.

Renal intercalated cells mediate secretion or absorption of Cl- and OH(-)/H(+) equivalents in the connecting segment (CNT) and cortical collecting duct (CCD). In so doing, they regulate acid-base balance, vascular volume and blood pressure. Cl(-) absorption is either electrogenic and amiloride-sensitive, or electroneutral and thiazide-sensitive. However, which Cl(-) transporter(s) are targeted by these diuretics is debated. While ENaC does not transport Cl(-), it modulates Cl(-) transport probably by generating a lumen-negative voltage, which drives Cl(-) flux across tight junctions. In addition, recent evidence indicates that ENaC inhibition increases electrogenic Cl- secretion via a type A intercalated cell. During ENaC blockade, Cl(-) is taken up across the basolateral membrane through the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and then secreted across the apical membrane through a conductive pathway (a Cl(-) channel or an electrogenic exchanger). the mechanism of this apical Cl(-) secretion is unresolved. In contrast, thiazide diuretics inhibit electroneutral Cl(-) absorption mediated by a Na(+)-dependent Cl(-)/HCO3(-) exchanger. The relative contribution of the thiazide and the amiloride-sensitive components of Cl(-) absorption varies between studies and probably depends on the treatment model employed. Cl(-) absorption increases markedly with angiotensin and aldosterone administration, largely by up-regulating the Na(+)-independent Cl(-)/HCO3(-) exchanger, pendrin. In the absence of pendrin (Slc26a4 (-/-) or pendrin null mice), aldosterone-stimulated Cl(-) absorption is significantly reduced, which attenuates the pressor response to this steroid hormone. Pendrin also modulates aldosterone-induced changes in ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function, at least in part, by altering luminal HCO3(-). This review summarizes mechanisms of Cl(-) transport in CNT and CCD, and how these transporters contribute to the regulation of extracellular volume and blood pressure.

BACKGROUND:

IKs channels, made of the pore-forming KCNQ1 and auxiliary KCNE1 subunits, play a key role in determining action potential duration (APD) in cardiac myocytes. The drug-induced KCNQ1 splice alteration remains unknown.

OBJECTIVE:

We study the modulation of KCNQ1 alternative splicing by amiloride and the consequent changes in IKs currents and action potentials (AP) in ventricular myocytes.

METHODS:

We study the modulation of KCNQ1 splicing by amiloride and the consequent changes in IKs currents and action potentials (AP) in ventricular myocytes. Canine endocardial, midmyocardial, and epicardial ventricular myocytes were isolated. Levels of KCNQ1a and KCNQ1b as well as a series of splicing factors were quantified by RT-PCR and Western blot. The impact of amiloride-induced alterations in KCNQ1b ratio on AP was measured using whole-cell patch clamp with and without isoproterenol.

RESULTS:

With 50 μmol/L amiloride for 6 hours, KCNQ1a at transcriptional and translational levels increases in midmyocardial but decreases in endo- and epicardial myocytes. Likewise, changes of splicing factors in midmyocardial were opposite to that in endo- and epicardial myocytes. In midmyocardial myocytes amiloride shortens APD and decreases isoproterenol-induced early afterdepolarizations significantly. The same amiloride-induced effects are demonstrated by using human ventricular model for action potentials simulations under β-adrenergic stimulation. Moreover, amiloride reduces the transmural dispersion of repolarization in pseudo-ECG.

CONCLUSIONS:

Amiloride regulates IKs currents and action potentials with transmural differences and reduces arrhythmogenecity through modulating KCNQ1 splicing. We suggested that modulation of KCNQ1 splicing may prevent arrhythmia.