Functional characterization of large conductance calcium-activated K + channel openers in bladder and vascular smooth muscle

Abstract

Calcium activated K(+) channels (K(Ca) channels) are found in a variety of smooth muscle tissues, the most characterized of which are the large conductance K(Ca) channels (BK(Ca) or maxi-K(+) channels). Recent medicinal chemistry efforts have identified novel BK(Ca) openers including 2-amino-5-(2-fluoro-phenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BMS-204352 and its analog 3-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-6-trifluoromethyl-1,3-dihydro-indol-2-one (compound 1), and 5,7-dichloro-4-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-1H-quinolin-2-one (compound 2). Although these compounds are effective BK(Ca) openers as shown by electrophysiological methods, little is known about their effects on smooth muscle contractility. In this study, the responsiveness of structurally diverse BK(Ca) openers-NS-8, compounds 1 and 2 and the well characterized nonselective NS-1619-was assessed using segments of endothelium denuded rat aorta, rat and guinea pig detrusor precontracted with extracellular K(+), and Landrace pig detrusor stimulated by electrical field. In all preparations, the compounds tested inhibited or completely abolished contractions with similar potencies (-logIC(50) values: 3.8 to 5.1). In rat aorta, in the presence of 80 mM K(+), the compounds significantly shifted the concentration-response curve to the right compared with those obtained in 30 mM K(+). These data are consistent with K(+) channel (BK(Ca) channel) activation as the underlying mechanism of relaxation by compounds that share the electrophysiological property of BK(Ca) current activation. The similar potencies at detrusor and vascular smooth muscle suggest that the achievement of smooth muscle selectivity in vitro with the representative compounds examined in this study may prove to be a challenge when targeting BK(Ca) channels for smooth muscle indications such as overactive bladder.

Ca2+‐activated K+ channel (KCa) stimulation improves relaxant capacity of PDE5 inhibitors in human penile arteries and recovers the reduced efficacy of PDE5 inhibition in diabetic erectile dysfunction

“…Although NS-8 is a known activator of BK (Parihar et al, 2003b;Malysz et al, 2004), NS1619 is considered to be the only selective BK opener by some authors (Ghatta et al, 2006) without any effect on KATP, voltage-gated K + , Na + or Ca 2+ channels (Olesen et al, 1994). According to previous results (Parihar et al, 2003b;Malysz et al, 2004), there are no qualitative differences between the effects induced by NS1619 and NS-8 in our experimental…”

“…In contrast, KCa activation potentiated the relaxant responses to PDE5 inhibition in HPRA, demonstrating tissue specificity. This potentiating effect is not limited to a pharmacological interaction of two specific molecules, since two different well-known activators of K Ca channels, NS1619 and NS-8 (Malysz et al, 2004) both potentiate sildenafil-induced relaxation. Similarly, vasodilatory responses to two different PDE5 inhibitors, sildenafil and tadalafil, are both enhanced by NS-8.…”

“…Sildenafil was a gift from Nitromed (Bedford, MA, USA) while tadalafil was provided by ICOS Corporation (Seattle, WA, USA). For in vitro experiments, all drugs were dissolved in deionized water, except for NS-8, NS1619 (Malysz et al, 2004), sildenafil (Angulo et al, 2010a) and tadalafil (Angulo et al, 2012), which were dissolved at 10 mM concentration in dimethylsulfoxide (DMSO). The subsequent dilutions were made in deionized water.…”

“…In ion efflux in vitro assays, EC50 for BK has reported to be in 1-4 mM range for NS1619 and NS-8 (Parihar et al, 2003b). Most data reporting the specificity of these molecules have been generated in functional studies where a dependency on BK activation in the NS-8-or NS1619-induced effects is abolished by BK-selective toxins (Holland et al, 1996;Malysz et al, 2004;Soder and Petkov, 2011). Although NS-8 is a known activator of BK (Parihar et al, 2003b;Malysz et al, 2004), NS1619 is considered to be the only selective BK opener by some authors (Ghatta et al, 2006) without any effect on KATP, voltage-gated K + , Na + or Ca 2+ channels (Olesen et al, 1994).…”

“…Most data reporting the specificity of these molecules have been generated in functional studies where a dependency on BK activation in the NS-8-or NS1619-induced effects is abolished by BK-selective toxins (Holland et al, 1996;Malysz et al, 2004;Soder and Petkov, 2011). Although NS-8 is a known activator of BK (Parihar et al, 2003b;Malysz et al, 2004), NS1619 is considered to be the only selective BK opener by some authors (Ghatta et al, 2006) without any effect on KATP, voltage-gated K + , Na + or Ca 2+ channels (Olesen et al, 1994). According to previous results (Parihar et al, 2003b;Malysz et al, 2004), there are no qualitative differences between the effects induced by NS1619 and NS-8 in our experimental…”