Functional expression of KCNQ (Kv7) channels in guinea pig bladder smooth muscle and their contribution to spontaneous activity.

BACKGROUND AND PURPOSE: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. EXPERIMENTAL APPROACH: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. KEY RESULTS: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca²âº-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20 µM) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. CONCLUSIONS AND IMPLICATIONS: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.

Comparison of mechanical and electrical activity and interstitial cells of Cajal in urinary bladders from wild-type and W/Wv mice.

BACKGROUND AND PURPOSE: W/W(v) and wild-type murine bladders were studied to determine whether the W/W(v) phenotype, which causes a reduction in, but not abolition of, tyrosine kinase activity, is a useful tool to study the function of bladder interstitial cells of Cajal (ICC). EXPERIMENTAL APPROACH: Immunohistochemistry, tension recordings and microelectrode recordings of membrane potential were performed on wild-type and mutant bladders. KEY RESULTS: Wild-type and W/W(v) detrusors contained c-Kit- and vimentin-immunopositive cells in comparable quantities, distribution and morphology. Electrical field stimulation evoked tetrodotoxin-sensitive contractions in wild-type and W/W(v) detrusor strips. Atropine reduced wild-type responses by 50% whereas a 25% reduction occurred in W/W(v) strips. The atropine-insensitive component was blocked by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both tissue types. Wild-type and W/W(v) detrusors had similar resting membrane potentials of -48 mV. Spontaneous electrical activity in both tissue types comprised action potentials and unitary potentials. Action potentials were nifedipine-sensitive whereas unitary potentials were not. Excitatory junction potentials were evoked by single pulses in both tissues. These were reduced by atropine in wild-type tissues but not in W/W(v) preparations. The atropine-insensitive component was abolished by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both preparations. CONCLUSIONS AND IMPLICATIONS: Bladders from W/W(v) mice contain c-Kit- and vimentin-immunopositive ICC. There are similarities in the electrical and contractile properties of W/W(v) and wild-type detrusors. However, significant differences were found in the pharmacology of the responses to neurogenic stimulation with an apparent up-regulation of the purinergic component. These findings indicate that the W/W(v) strain may not be the best model to study ICC function in the bladder.

Characterization of T-type calcium current and its contribution to electrical activity in rabbit urethra.

Rabbit urethral smooth muscle cells were studied at 37 degrees C by using the amphotericin B perforated-patch configuration of the patch-clamp technique, using Cs(+)-rich pipette solutions. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca(2+) currents, were recorded. Fitting steady-state inactivation curves for the L current with a Boltzmann equation yielded a V(1/2) of -41 +/- 3 mV. In contrast, the T current inactivated with a V(1/2) of -76 +/- 2 mV. The L currents were reduced by nifedipine (IC(50) = 225 +/- 84 nM), Ni(2+) (IC(50) = 324 +/- 74 microM), and mibefradil (IC(50) = 2.6 +/- 1.1 microM) but were enhanced when external Ca(2+) was substituted with Ba(2+). The T current was little affected by nifedipine at concentrations <300 nM but was increased in amplitude when external Ca(2+) was substituted with Ba(2+). Both Ni(2+) and mibefradil reduced the T current with an IC(50) = 7 +/- 1 microM and approximately 40 nM, respectively. Spontaneous electrical activity recorded with intracellular electrodes from strips of rabbit urethra consisted of complexes comprising a series of spikes superimposed on a slow spontaneous depolarization (SD). Inhibition of T current reduced the frequency of these SDs but had no effect on either the number of spikes per complex or the amplitude of the spikes. In contrast, application of nifedipine failed to significantly alter the frequency of the SD but reduced the number and amplitude of the spikes in each complex.