brl-37344 and iberiotoxin

To test if carbachol (CCh)-evoked Ca. Isometric tension recordings were made from strips of murine detrusor, and intracellular Ca. The RT-PCR and qPCR experiments showed that β1-, β2- and β3-AR were expressed in murine detrusor, but that β3-ARs were the most abundant sub-type. The selective β3-AR agonist BRL37344 reduced the amplitude of CCh-induced contractions of detrusor smooth muscle. These responses were unaffected by addition of the BK channel blocker iberiotoxin. BRL37344 also reduced the amplitude of CCh-induced Ca. Inhibition of cholinergic-mediated contractions of the detrusor by β3-AR agonists was associated with a reduction in Ca

Topics: Adrenergic beta-3 Receptor Agonists; Adrenergic beta-Agonists; Animals; Calcium; Carbachol; Cholinergic Agonists; Ethanolamines; Female; Male; Mice, Inbred C57BL; Muscle Cells; Muscle Contraction; Muscle, Smooth; Peptides; Urinary Bladder

The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is a major regulator of detrusor smooth muscle (DSM) contractility thus facilitating urinary bladder function. Recent findings suggest that activation of β3-adrenoceptors causes DSM relaxation. However, it is unknown whether the β3-adrenoceptor-mediated DSM relaxation is BK channel-dependent during nerve-evoked contractions. To test this hypothesis, we induced nerve-evoked contractions in rat DSM isolated strips by using a tissue bath system equipped with platinum electrodes for electrical field stimulation (EFS). (±)-(R(*),R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL37344), a β3-adrenoceptor agonist, significantly decreased the amplitude and muscle force of the 20 Hz EFS-induced DSM contractions in a concentration-dependent manner. The BRL37344 inhibitory effect was significantly antagonized by 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A), a β3-adrenoceptor antagonist. We further isolated the cholinergic from the purinergic component of the 0.5-50 Hz EFS-induced DSM contractions by using selective inhibitors, atropine as well as suramin and α,β-methylene-ATP. We found that BRL37344 inhibited both the purinergic and cholinergic components of the nerve-evoked contractions in rat DSM isolated strips. The pharmacological blockade of the BK channels with iberiotoxin, a selective BK channel inhibitor, increased the amplitude and muscle force of the 20 Hz EFS-induced contractions in rat DSM isolated strips. In the presence of iberiotoxin, there was a significant reduction in the BRL37344-induced inhibition of the 20 Hz EFS-induced contractions in rat DSM isolated strips. These latter findings suggest that BK channels play a critical role in the β3-adrenoceptor-mediated inhibition of rat DSM nerve-evoked contractions.

Topics: Acetylcholine; Adrenergic beta-3 Receptor Agonists; Adrenergic beta-3 Receptor Antagonists; Animals; Electric Stimulation; Ethanolamines; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Male; Muscle Relaxation; Muscle, Smooth; Peptides; Potassium Channel Blockers; Propanolamines; Purines; Rats; Receptors, Adrenergic, beta-3; Urinary Bladder

To investigate the mechanism by which BRL37344, a β3-adrenergic receptor (β3-ARs) agonist, facilitates the inhibition of nerve-evoked contractions in human detrusor smooth muscle (DSM) isolated strips and to identify the role of large-conductance Ca(2+)-activated K(+) (BK) channels in this process.. Human DSM specimens were obtained from open bladder surgeries on patients without preoperative history of overactive bladder symptoms. Isometric DSM tension recordings were conducted using force-displacement transducers and thermostatically controlled tissue baths. Nerve-evoked contractions were generated by electrical field stimulation (EFS).. BRL37344, a β3-AR agonist, significantly decreased the amplitude, muscle force, and duration of the DSM contractions induced by 20 Hz EFS, in a concentration-dependent manner. This BRL37344-mediated inhibition of the amplitude and muscle force of the nerve-evoked DSM contraction was significantly reduced by iberiotoxin, a highly selective inhibitor of the BK channel, revealing a role for BK channels in the β3-AR-induced inhibition of human DSM nerve-evoked contractions. We further used atropine, α,β-methylene-ATP, and suramin to separate the cholinergic and purinergic components of human DSM nerve-evoked contractions. We found that the β3-AR agonist, BRL37344, inhibited both components of the EFS-induced (0.5-50 Hz) DSM contractions.. This study supports the concept that β3-AR agonists inhibit nerve-evoked contractions in human DSM. We have further revealed that BK channels play a critical role in BRL37344-mediated relaxation of nerve-evoked contractions in human DSM. The study suggests that in addition to β3-ARs, BK channels may also represent promising pharmacologic targets in the treatment of urinary bladder dysfunction.

Topics: Adenosine Triphosphate; Adrenergic beta-3 Receptor Agonists; Aged; Atropine; Dose-Response Relationship, Drug; Electric Stimulation; Ethanolamines; Female; Humans; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Male; Middle Aged; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Peptides; Potassium Channel Blockers; Purinergic Agonists; Purinergic Antagonists; Suramin; Urinary Bladder

We examined the effects of beta-adrenoceptor agonists on the membrane currents of smooth muscle cells from the human urinary bladder using a whole-cell patch clamp to investigate the involvement of Ca(2+)-activated K(+) (K(Ca)) channels in relaxation by beta-adrenergic agonists. With 0.05 mmol/l EGTA in the patch pipette, depolarizing pulses evoked outward rectifying currents. Isoproterenol (1 micromol/l) significantly increased the membrane currents by 75% at +80 mV with 0.05 mmol/l EGTA pipette solution. BRL 37344 (1 micromol/l) significantly increased the membrane currents by 44% at +80 mV. Iberiotoxin (100 nmol/l) significantly decreased the membrane currents by 60% at +80 mV. In the presence of iberiotoxin, the potentiation of the outward currents by isoproterenol was greatly suppressed and, in the presence of iberiotoxin and apamin (1 micromol/l), the potentiation by isoproterenol was totally abolished. On the other hand, with 5 mmol/l EGTA pipette solution, depolarizing pulses evoked smaller outward currents. Isoproterenol (1 micromol/l) did not change the membrane currents with 5 mmol/l EGTA pipette solution. The real-time PCR analysis revealed the expression of beta(2)-adrenoceptors in the cells. These results suggest that Ca(2+)-activated and iberiotoxin- and apamin-sensitive currents via both large-conductance and small-conductance K(Ca) channels could be increased by stimulation of beta(2)-adrenoceptors.

Topics: Adrenergic beta-Agonists; Apamin; Cell Line; Electrophysiology; Ethanolamines; Gene Expression Regulation; Humans; Isoproterenol; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Muscle, Smooth; Patch-Clamp Techniques; Peptides; Receptors, Adrenergic, beta; Small-Conductance Calcium-Activated Potassium Channels; Urinary Bladder

Beta3-adrenoreceptor modulation in human myometrium during pregnancy is linked functionally to myometrial inhibition. Maxi-K+ channels (BK(Ca)) play a significant role in modulating cell membrane potential and excitability.. This study was designed to investigate the potential involvement of BK(Ca) channel function in the response of human myometrium to beta3-adrenoceptor activation.. Single and whole-cell electrophysiological BK(Ca) channel recordings from freshly dispersed myocytes were obtained in the presence and absence of BRL37344, a specific beta3-adrenoreceptor agonist. The in vitro effects of BRL37344 on isolated myometrial contractions, in the presence and absence of the specific BK(Ca) channel blocker, iberiotoxin (IbTX), were investigated.. The study was carried out at the Clinical Science Institute.. Myometrial biopsies were obtained at elective cesarean delivery.. No intervention was applied.. Open state probability of single channel recordings, whole cell currents, and myometrial contractile activity were measured.. Single-channel recordings identified the BK(Ca) channel as a target of BRL37344. BRL37344 significantly increased the open state probability of this channel in a concentration-dependent manner (control 0.031 +/- 0.004; 50 microM BRL37344 0.073 +/- 0.005 (P < 0.001); and 100 microM BRL37344 0.101 +/- 0.005 (P < 0.001). This effect was completely blocked after preincubation of the cells with 1 microM bupranolol, a nonspecific beta-adrenoreceptor blocker, or 100 nM SR59230a, a specific beta3-adrenoreceptor antagonist. In addition, BRL37344 increased whole-cell currents over a range of membrane potentials, and this effect was reversed by 100 nM IbTX. In vitro isometric tension studies demonstrated that BRL37344 exerted a significant concentration-dependent relaxant effect on human myometrial tissue (P < 0.05), and preincubation of these strips with IbTX attenuated this effect on both spontaneous and oxytocin-induced contractions (44.44 and 57.84% at 10(-5) M, respectively).. These findings outline that activation of the BK(Ca) channel may explain the potent uterorelaxant effect of beta3-adrenoreceptor agonists.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Bupranolol; Dose-Response Relationship, Drug; Electrophysiology; Ethanolamines; Female; Humans; In Vitro Techniques; Isometric Contraction; Membrane Potentials; Muscle Cells; Muscle Relaxation; Myometrium; Oxytocin; Peptides; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Propanolamines; Receptors, Adrenergic, beta-3; Uterine Contraction; Uterus