Spontaneous and neurally activated depolarizations in smooth muscle cells of the guinea-pig urethra

Abstract

1. Membrane potential recordings were made from longitudinal smooth muscle cells of the guinea-pig urethra using conventional microelectrode techniques. 2. Smooth muscle cells of the urethra developed spontaneous transient depolarizations (STDs) and slow waves. Single unit STDs had amplitudes of approximately 5 mV and slow waves seemed to occur as amplitude multiples of single unit STDs. 3. STDs and slow waves were abolished by niflumic acid or low chloride solution and also by cyclopiazonic acid (CPA), BAPTA or high concentrations of caffeine. Lower concentrations of caffeine abolished slow waves but not STDs. Nifedipine inhibited slow waves but not STDs. 4. When stochastic properties of STDs were examined, it was found that the intervals between occurrences were not well modelled by Poisson statistics, instead the STDs appeared to be clustered. 5. Transmural stimulation evoked excitatory junctional potentials (EJPs) and triggered slow waves which were abolished by either alpha,beta-methylene-ATP or tetrodotoxin. Evoked slow waves were also abolished by caffeine, co-application of caffeine and ryanodine or by CPA which left EJPs unaffected. 6. In conclusion, smooth muscle cells of urethra exhibit STDs which are clustered rather than random events, and are the result of spontaneous Ca2+ release from intracellular stores and subsequent activation of Ca2+-activated chloride channels. STDs sum to activate L-type Ca2+ channels which contribute to the sustained phase of slow waves. Stimulation of purinoceptors by neurally released ATP initiates EJPs and also causes the release of Ca2+ from intracellular stores to evoke slow waves.

Figure 1. Effects of nifedipine on spontaneous slow waves and STDs

Aa, slow waves (indicated by arrowheads in each panel) and STDs (indicated by arrows in each panel) were recorded in control conditions. Ab, nifedipine (1 μm) reduced the duration of slow waves without obvious changes to their amplitude or frequency. STDs persisted in the presence of 1 μm nifedipine. B, nifedipine (1 μm) reduced the duration, but not the amplitude or rise time, of slow waves. Ca, slow waves and STDs were recorded from separate preparations in control conditions. Cb, nifedipine (1 μm) reduced not only the duration but also the amplitude of slow waves. STDs persisted in the presence of 1 μm nifedipine. D, nifedipine (1 μm) reduced not only the duration but also the amplitude of slow waves without reducing their rise times. A and B were recorded in one preparation, C and D in another. RMP, resting membrane potential.

Figure 2. Effects of low chloride solution and niflumic acid on slow waves and STDs

Aa, slow waves and STDs were recorded in control conditions. Ab, low chloride solution abolished both slow waves and STDs. Ba, in another preparation slow waves and STDs were recorded in control conditions. Bb, niflumic acid (50 μm) abolished slow waves and STDs. The scale bar on the right refers to both traces. RMP, resting membrane potential.

Figure 6. Effects of tetrodotoxin and nifedipine on responses produced by transmural nerve stimulation

Aa, trains of impulses (supramaximal currents, 100 μs, 1 Hz, 5 s) initiated EJPs and triggered a slow wave at the third impulse. Ab, both EJPs and the evoked slow wave were abolished by 1 μm tetrodotoxin (TTX). Ba, trains of impulses (supramaximal currents, 100 μs, 10 Hz, 1 s) invariably evoked a slow wave which was abolished by 1 μm tetrodotoxin (Bb). An evoked slow wave (Cb) had similar amplitude and time course to a spontaneous slow wave (Ca). Da, nifedipine (1 μm) reduced the duration of the slow wave without reducing its amplitude or rise time (Db). A and B were recorded from one preparation; C and D were recorded from 2 different preparations. The scale bars in B also refer to A. The scale bars in D also refer to C. RMP, resting membrane potential.

Figure 3. Effects of caffeine, CPA and BAPTA on slow waves and STDs

Aa, slow waves and STDs were recorded in the presence of nifedipine (1 μm). Ab, caffeine (1 mM) abolished slow waves but not STDs. Ba, slow waves and STDs were recorded from a separate preparation in control conditions. Bb, caffeine (10 mM) abolished both slow waves and STDs. Ca, slow waves and STDs were recorded in the presence of nifedipine (1 μm). Cb, CPA (10 μm) abolished both slow waves and STDs. Da, slow waves and STDs were recorded in the presence of nifedipine (1 μm). Db, BAPTA (50 μm) abolished both slow waves and STDs. A, C and D were recorded in one preparation; B was recorded in another. The scale bars on the right refer to all traces. RMP, resting membrane potential.

Figure 4. Summation of single unit STDs

Aa, a single unit STD was recorded in the presence of nifedipine (1 μm). Ab, summation of 2 units. Ac, summation of 5 units. The superimposed dotted trace indicates a single unit. Ba, in a separate preparation, a single unit STD was recorded in the presence of 1 μm nifedipine. Bb, summation of 3 units. The superimposed dotted trace again indicates a single unit. Bc, summation of 5 units. A and B were recorded from 2 different tissues. The scale bars on the right refer to all traces. The horizontal lines indicate amplitudes of single unit STDs in each trace.

Figure 5. ln(survivor) curves plotted from the data obtained from urethral smooth muscle preparations

A, the empirical ln(survivor) curve plotted for the whole range of intervals obtained from a preparation which exhibited STDs but not slow waves. B, the empirical ln(survivor) curve plotted for the partial range of intervals obtained from a preparation which exhibited both slow waves and STDs. All data were obtained in the presence of 1 μm nifedipine. In each case, the straight line represents the ln(survivor) curve predicted for a Poisson process of random and statistically independent events of mean frequency equal to that recorded. The empirical curves lie below the Poisson prediction for short intervals, indicating that there is an excess of closely spaced events. A, cell I of Table 1; B, cell VII of Table 1.

Figure 7. Effects of phenylephrine, α,β-methylene-ATP and guanethidine on slow waves

A, phenylephrine (1 μm) increased the frequency of spontaneous slow waves and caused a sustained depolarization. B, α,β-methylene-ATP (10 μm) caused a transient increase in the frequency of spontaneous slow waves and a transient depolarization. Ca, an averaged slow wave recorded in the presence of 1 μm nifedipine. Cb, guanethidine (10 μm) did not effect the evoked slow wave. Cc, α,β-methylene-ATP (10 μm) abolished both the evoked slow wave and EJPs. Each trace in C is an average of 3-5 recordings. The scale bars in B also refer to A. The scale bars in C refer to all traces in C. RMP, resting membrane potential.

Figure 8. Effects of caffeine, caffeine plus ryanodine and CPA on evoked slow waves

Aa, in the presence of 1 μm nifedipine, slow waves were evoked by trains of impulses (supramaximal currents, 100 μs, 20 Hz, 0.25 s). Ab, caffeine (3 mM) abolished slow waves leaving residual responses which appeared to be summed EJPs. Ba, in the presence of nifedipine (1 μm) slow waves were evoked by similar trains of impulses. Bb, co-application of caffeine (3 mM) and ryanodine (10 μm) irreversibly abolished evoked slow waves and left summed EJPs after 1 h washout. Cb, CPA (10 μm) abolished evoked slow waves, again leaving summed EJPs. All traces were recorded in the same preparation and each trace was an average of 3-5 recordings. The scale bars on the right refer to all traces. RMP, resting membrane potential.