Stimulation of (cid:1) 3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca 2 (cid:2) -activated K (cid:2) channels

Stimulation of (cid:1) 3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca 2 (cid:2) -activated K (cid:2) channels. Am J Physiol Cell Physiol 295: C1344–C1353, 2008. 2008; doi:10.1152/ajpcell.00001. 2008. —We investigated the role of large-conductance Ca 2 (cid:2) -activated K (cid:2) (BK) channels in (cid:1) 3-adrenoceptor ( (cid:1) 3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a speciﬁc (cid:1) 3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a speciﬁc (cid:1) 3-AR antagonist, and H89, a PKA inhibitor, reduced the inhibitory effect of BRL 37344. Iberiotoxin, a speciﬁc BK channel inhibitor, shifts the BRL 37344 concentration response curves for contraction amplitude, net muscle force, and tone to the right. Freshly dispersed UBSM cells and the perforated mode of the patch-clamp technique were used to determine further the role of (cid:1) 3-AR stimulation by BRL 37344 on BK channel activity. BRL 37344 increased spontaneous, transient, outward BK current (STOC) frequency by 46.0 (cid:3) 20.1%. In whole cell mode at a holding potential of V h (cid:4) 0 mV, the single BK channel amplitude was 5.17 (cid:3) 0.28 pA, whereas in the presence of BRL 37344, it was 5.55 (cid:3) 0.41 pA. The BK channel open probability was also unchanged. In the presence of ryanodine and nifedipine, the current-voltage relationship in response to depolarization steps in the presence and absence of BRL 37344 was identical. In current-clamp mode, BRL 37344 caused membrane potential hyperpolarization from (cid:5) 26.1 (cid:3) 2.1 mV (control) to (cid:5) 29.0 (cid:3) 2.2 mV. The BRL 37344-induced hyperpolarization was eliminated by application of iberiotoxin, tetraethylammonium or ryanodine. The data indicate that stimulation of (cid:1) 3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation. the cell capacitance (measured as NP o /pF). In some cells, at the end of the recordings, IBTX (200 nM) was applied to conﬁrm that the recorded currents were through BK channels. UBSM cell membrane potential was recorded using the current-clamp mode of the patch-clamp technique. All patch-clamp experiments were carried out at room temperature (22–23°C). tiﬁc. Data analysis and statistics. UBSM contraction data were analyzed using MiniAnalysis (Synaptosoft). This software has allowed us to analyze the changes in all four major parameters of the UBSM phasic and tonic contractions (tone)-phasic contraction amplitude, phasic contractions frequency, net muscle force, and muscle tone. Data were further analyzed with GraphPad Prism (version 4) and presented using CorelDraw Graphic Suite X3 (Corel) software. Cumulative concentration response curves for BRL 37344 were obtained by adding increasing concentrations of the drugs directly to the tissue baths every 10 min. A 3-min period from the 7th to the 10th min after exposure to each drug concentration was taken as the analysis period. To compare the phasic contractions parameters, data were normalized to the spontaneous contractions and expressed as percentages. Net muscle force (muscle force integral) was determined by integrating the area under the curve of the phasic contractions component. Tone was determined by measuring changes of the phasic contraction baseline curve. Patch-clamp electrophysiological data were analyzed with Clampﬁt version 10.1. Summary data are presented as means (cid:3) SE for n , the number of separate UBSM strips or single cells, respectively, and N , the number of individual rats. The data were assessed for statistical signiﬁcance using the one-tailed paired Student’s t -test. A P value (cid:10) 0.05 was considered signiﬁcant.