RESUMO
High-conductance calcium-activated potassium (Maxi-K) channels are present in smooth muscle where they regulate tone. Activation of Maxi-K channels causes smooth muscle hyperpolarization and shortening of action-potential duration, which would limit calcium entry through voltage-dependent calcium channels leading to relaxation. Although Maxi-K channels appear to indirectly mediate the relaxant effects of a number of agents, activators that bind directly to the channel with appropriate potency and pharmacological properties useful for proof-of-concept studies are not available. Most agents identified to date display significant polypharmacy that severely compromises interpretation of experimental data. In the present study, a high-throughput, functional, cell-based assay for identifying Maxi-K channel agonists was established and used to screen a large sample collection (>1.6 million compounds). On the basis of potency and selectivity, a family of tetrahydroquinolines was further characterized. Medicinal chemistry efforts afforded identification of compound X, from which its two enantiomers, Y and Z, were resolved. In in vitro assays, Z is more potent than Y as a channel activator. The same profile is observed in tissues where the ability of either agent to relax precontracted smooth muscles, via a potassium channel-dependent mechanism, is demonstrated. These data, taken together, suggest that direct activation of Maxi-K channels represents a mechanism to be explored for the potential treatment of a number of diseases associated with smooth muscle hyperexcitability.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Músculo Liso/fisiologia , Animais , Células CHO , Cromatografia Líquida , Cricetinae , Cricetulus , Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Relaxamento MuscularRESUMO
1 A constant intraluminal pressure system was used to evaluate the effects of Kv1 channel blockers on the peristaltic activity of guinea-pig ileum. 2 The nortriterpene correolide, a non-selective inhibitor of all Kv1 sub-types, causes progressive and sustained reduction of the pressure threshold for eliciting peristaltic contractions. 3 Margatoxin (MgTX), alpha-dendrotoxin (alpha-DTX) and dendrotoxin-K (DTX-K), highly selective peptidyl inhibitors of certain Kv1 sub-types, cause immediate reduction of the pressure threshold. This effect subsides with time, irrespective of the peptides' concentration in the bath. In preparations pretreated with saturating concentrations of MgTX, correolide further stimulates the peristaltic activity. 4 Iberiotoxin (IbTX), a selective inhibitor of the high-conductance Ca(2+)-activated K(+) (BK) channels, and charybdotoxin (ChTX), which inhibits Kv1.2 and Kv1.3 as well as BK channels, fail to stimulate the peristaltic activity. 5 Blockade of muscarinic receptors by atropine reduces, and occasionally suppresses the peristaltic activity of guinea-pig ileum. In atropine-treated preparations, correolide and MgTX retain their abilities to reduce the pressure threshold and are able to restore the peristaltic reflex in the preparations where this reflex was suppressed by atropine. 6 The stimulatory effect of correolide and MgTX in atropine-treated preparations is abolished by subsequent addition of selective antagonists of both NK1 and NK2 receptors. 7 In conclusion, blockade of Kv1, particularly Kv1.1 channels, increases the peristaltic activity of guinea-pig ileum by enhancing the release of neurotransmitters at the enteric nervous system. In contrast, stimulation of the myogenic motility by blockade of BK channels does not affect the threshold for the peristaltic reflex.