The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, β(1)-, and β(4)-subunits. Western blot and immunocytochemistry confirmed BK channel α, β(1), and β(4) protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K(+) current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.

GV. ␤-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca 2ϩ -activated K ϩ channel. Am J Physiol Renal Physiol 295: F1149 -F1157, 2008. First published August 13, 2008; doi:10.1152/ajprenal.00440.2007.-In urinary bladder smooth muscle (UBSM), stimulation of ␤-adrenergic receptors (␤-ARs) leads to activation of the large-conductance Ca 2ϩ -activated K ϩ (BK) channel currents (Petkov GV and Nelson MT. Am J Physiol Cell Physiol 288: C1255-C1263, 2005. In this study we tested the hypothesis that the BK channel mediates UBSM relaxation in response to ␤-AR stimulation using the highly specific BK channel inhibitor iberiotoxin (IBTX) and a BK channel knockout (BK-KO) mouse model in which the gene for the pore-forming subunit was deleted. UBSM strips isolated from wild-type (WT) and BK-KO mice were stimulated with 20 mM K ϩ or 1 M carbachol to induce phasic and tonic contractions. BK-KO and WT UBSM strips pretreated with IBTX had increased overall contractility, and UBSM BK-KO cells were depolarized with ϳ12 mV. Isoproterenol, a nonspecific ␤-AR agonist, and forskolin, an adenylate cyclase activator, decreased phasic and tonic contractions of WT UBSM strips in a concentrationdependent manner. In the presence of IBTX, the concentrationresponse curves to isoproterenol and forskolin were shifted to the right in WT UBSM strips. Isoproterenol-and forskolin-mediated relaxations were enhanced in BK-KO UBSM strips, and a leftward shift in the concentration-response curves was observed. The leftward shift was eliminated upon PKA inhibition with H-89, suggesting upregulation of the ␤-AR-cAMP pathway in BK-KO mice. These results indicate that the BK channel is a key modulator in ␤-AR-mediated relaxation of UBSM and further suggest that alterations in BK channel expression or function could contribute to some pathophysiological conditions such as overactive bladder and urinary incontinence.BK channel knockout mouse; isoproterenol; forskolin; iberiotoxin DURING VOIDING, the urinary bladder smooth muscle (UBSM) contracts forcefully to expel urine. UBSM exhibits spontaneous action potentials that are associated with the phasic nature of the contractions in this tissue (2,6,17). Ca 2ϩ entry through dihydropyridine-sensitive L-type voltage-gated Ca 2ϩ (Ca V ) channels is responsible for the upstroke of the action potential and leads to an increase in global intracellular Ca 2ϩ , which activates the UBSM phasic contractions. Blocking L-type Ca V channels eliminates the spontaneous action potentials and contractions in UBSM (2, 6). The repolarization phase of the UBSM action potential is mediated by the activity of the large-conductance Ca 2ϩ -activated K ϩ (BK) channels (6) and perhaps the voltage-gated K ϩ (K V ) channels (23). Pharmacological inhibition of UBSM BK channels with a highly specific inhibitor, iberiotoxin (IBTX), increases the action potential duration and frequency, causes membrane potential depolarization (6, 19), and increases the amplitude of phasic contractions (8, 17)...

BackgroundSea cucumbers (Holothuroidea; Echinodermata) have the capacity to regenerate lost tissues and organs. Although the histological and cytological aspects of intestine regeneration have been extensively studied, little is known of the genetic mechanisms involved. There has, however, been a renewed effort to develop a database of Expressed Sequence Tags (ESTs) in Apostichopus japonicus, an economically-important species that occurs in China. This is important for studies on genetic breeding, molecular markers and special physiological phenomena. We have also constructed a library of ESTs obtained from the regenerative body wall and intestine of A. japonicus. The database has increased to ∼30000 ESTs.ResultsWe used RNA-Seq to determine gene expression profiles associated with intestinal regeneration in A. japonicus at 3, 7, 14 and 21 days post evisceration (dpe). This was compared to profiles obtained from a normally-functioning intestine. Approximately 5 million (M) reads were sequenced in every library. Over 2400 up-regulated genes (>10%) and over 1000 down-regulated genes (∼5%) were observed at 3 and 7dpe (log2Ratio≥1, FDR≤0.001). Specific “Go terms” revealed that the DEGs (Differentially Expressed Genes) performed an important function at every regeneration stage. Besides some expected pathways (for example, Ribosome and Spliceosome pathway term), the “Notch signaling pathway,” the “ECM-receptor interaction” and the “Cytokine-cytokine receptor interaction” were significantly enriched. We also investigated the expression profiles of developmental genes, ECM-associated genes and Cytoskeletal genes. Twenty of the most important differentially expressed genes (DEGs) were verified by Real-time PCR, which resulted in a trend concordance of almost 100% between the two techniques.ConclusionOur studies demonstrated dynamic changes in global gene expression during intestine regeneration and presented a series of candidate genes and enriched pathways that contribute to intestine regeneration in sea cucumbers. This provides a foundation for future studies on the genetics/molecular mechanisms associated with intestine regeneration.

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