Opening Of Large-conductance Ca Research Articles

Potassium and ANO1/ TMEM16A chloride channel profiles distinguish atypical and typical smooth muscle cells from interstitial cells in the mouse renal pelvis

Although atypical smooth muscle cells (SMCs) in the proximal renal pelvis are thought to generate the pacemaker signals that drive pyeloureteric peristalsis, their location and electrical properties remain obscure. Standard patch clamp, intracellular microelectrode and immunohistochemistry techniques were used. To unequivocally identify SMCs, transgenic mice with enhanced yellow fluorescent protein (eYFP) expressed in cells containing α-smooth muscle actin (α-SMA) were sometimes used. Atypical SMCs were distinguished from typical SMCs by the absence of both a transient 4-aminopyridine-sensitive K(+) current (I(KA) ) and spontaneous transient outward currents (STOCs) upon the opening of large-conductance Ca(2+) -activated K(+) (BK) channels. Many typical SMCs displayed a slowly activating, slowly decaying Cl(-) current blocked by niflumic acid (NFA). Immunostaining for K(V) 4.3 and ANO1/ TMEM16A Cl(-) channel subunits co-localized with α-SMA immunoreactive product predominately in the distal renal pelvis. Atypical SMCs fired spontaneous inward currents that were either selective for Cl(-) and blocked by NFA, or cation-selective and blocked by La(3+) . α-SMA(-) interstitial cells (ICs) were distinguished by the presence of a Xe991-sensitive K(V) 7 current, BK channel STOCs and Cl(-) selective, NFA-sensitive spontaneous transient inward currents (STICs). Intense ANO1/ TMEM16A and K(V) 7.5 immunostaining was present in Kit(-) α-SMA(-) ICs in the suburothelial and adventitial regions of the renal pelvis. We conclude that K(V) 4.3(+) α-SMA(+) SMCs are typical SMCs that facilitate muscle wall contraction, that ANO1/ TMEM16A and K(V) 7.5 immunoreactivity may be selective markers of Kit(-) ICs and that atypical SMCs which discharge spontaneous inward currents are the pelviureteric pacemakers.

Molecular Mechanisms for Large Conductance Ca2+-Activated K+ Channel Activation by a Novel Opener, 12,14-Dichlorodehydroabietic Acid

Our recent study has revealed that 12,14-dichlorodehydroabietic acid (diCl-DHAA), which is synthetically derived from a natural product, abietic acid, is a potent opener of large conductance Ca(2+)-activated K(+) (BK) channel. Here, we examined, by using a channel expression system in human embryonic kidney 293 cells, the mechanisms underlying the BK channel opening action of diCl-DHAA and which subunit of the BK channel (alpha or beta1) is the site of action for diCl-DHAA. BK channel activity was significantly enhanced by diCl-DHAA at concentrations of 0.1 microM and higher in a concentration-dependent manner. diCl-DHAA enhanced the activity of BKalpha by increasing sensitivity to both Ca(2+) and membrane potential without changing the single channel conductance. It is notable that the increase in BK channel open probability by diCl-DHAA showed significant inverse voltage dependence, i.e., larger potentiation at lower potentials. Since coexpression of beta1 subunit with BKalpha did not affect the potency of diCl-DHAA, the site of action for diCl-DHAA is suggested to be BKalpha subunit. Moreover, kinetic analysis of single channel currents indicates that diCl-DHAA opens BKalpha mainly by decreasing the time staying in a long closed state. Although reconstituted voltage-dependent Ca(2+) channel current was significantly reduced by 1 microM diCl-DHAA, BK channels were selectively activated at lower concentrations. These results indicate that diCl-DHAA is one of the most potent BK channel openers acting on BKalpha and a useful prototype compound to develop a novel BK channel opener.

Involvement of guanylyl cyclase, protein kinase A and Na +K + ATPase in relaxations of bovine isolated bronchioles induced by GEA 3175, an NO donor

The present study was designed to investigate the role of the sodium potassium adenosine triphosphatase (the Na +K + ATPase) in relaxation of bovine isolated bronchioles by a new NO donor, GEA 3175 (3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulphonyl]amino]-)hydroxide)). Bronchioles were mounted in a wire myograph for isometric tension recordings and contracted with 5-hydroxytryptamine (5-HT) or a K + rich solution. Concentration-dependent relaxations evoked by GEA 3175 were inhibited by ouabain or K + free solution. The guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 μM) and ouabain (10 nM) reduced GEA 3175-evoked relaxations to the same extent without any additive effect. Iberiotoxin (10 nM), an inhibitor of large conductance Ca 2+-activated K + channels inhibited GEA 3175-evoked relaxations to the same extent as ouabain. Combining ouabain and iberiotoxin completely abolished GEA 3175 relaxation. An inhibitor of protein kinase G (PKG), Rp-β-phenyl-1,N 2-etheno-8-bromo-guanosine-3′-5′-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPs), slightly reduced GEA 3175-induced relaxations. An inhibitor of cyclic AMP-dependent kinase (PKA), Rp-adenosine-3′-5′-cyclic phosphorothioate (Rp-cAMPs), inhibited the GEA 3175-induced relaxations to the same extent as ouabain. Inhibition of both PKG and PKA abolished GEA 3175 relaxation. The study provides evidence that the NO donor GEA 3175 causes guanylyl cyclase-dependent relaxations, taking place through cyclic GMP and cyclic AMP-dependent protein kinases followed by opening of large conductance Ca 2+-activated K + channels and activation of smooth muscle Na +K + ATPase.

Effect of 4-(5-Chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl)-6-(trifluoromethyl)-quinolin-2(1H)-one (BMS-223131), a Novel Opener of Large Conductance Ca2+-Activated K+(Maxi-K) Channels on Normal and Stress-Aggravated Colonic Motility and Visceral Nociception

We evaluated the effects of 4-(5-chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl)-6-(trifluoromethyl)-quinolin-2(1H)-one (BMS-223131), an opener of large conductance Ca(2+)-activated potassium (maxi-K) channels, on normal and stress-exacerbated colonic motility and visceral nociception in the rat. Fecal output was employed as an index of motility. Visceral nociception, in response to intracolonic balloon distension (10-90 mm Hg; 30 s duration), was evaluated using one of three indices: change in blood pressure, abdominal withdrawal, or myoelectrical activity. BMS-223131 (2, 6, or 20 mg/kg i.p.) produced a small but dose-dependent and significant reduction in cumulative 24-h fecal output. Fecal output in response to stress (1-h restraint plus bursts of air to the face) was markedly inhibited by BMS-223131, and moisture content was significantly reduced. With regard to visceral pain, the transient and distention-dependent reduction in arterial pressure in anesthetized animals was inhibited by BMS-223131 in a dose-dependent manner. Distension-induced abdominal withdrawal in conscious rats was also dose-dependently attenuated by BMS-223131. BMS-223131 at a dose of 20 mg/kg markedly attenuated the increase in myoelectrical activity evoked by balloon distention in conscious animals. BMS-223131 was also evaluated in viscerally hypersensitive rats (sensitized as neonates by intracolonic mustard oil) where it produced a robust dose-dependent attenuation of the abdominal withdrawal response. Compared with naive animals, BMS-223131 was more potent in the sensitized animals. Thus, BMS-223131 effectively reduced stress-induced colonic motility and visceral nociception supporting the potential utility of maxi-K channel openers for the treatment of bowel disorders involving dysfunctional motility and visceral sensitivity.