Correlation between spontaneous electrical, calcium and mechanical activity in detrusor smooth muscle of the guinea-pig bladder

Abstract

1. To investigate the cellular mechanisms underlying spontaneous excitation of smooth muscle of the guinea-pig urinary bladder, isometric tension was measured in muscle bundles while recording the membrane potential from a cell in the bundle with a microeletrode. Changes in the intracellular calcium concentration ([Ca(2+)](i); calcium transients) were recorded in strips loaded with the fluorescent dye, fura-PE3. 2. In 40% of preparations, individual action potentials and contractions, which were abolished by nifedipine (1 microm), were generated. In the remaining preparations, bursting action potentials and contractions were generated. Contractions were again abolished by nifedipine (1 microm), while higher concentrations of nifedipine (10-30 microm) were required to prevent the electrical activity. 3. Carbachol (0.1 microm) increased the frequency of action potentials and corresponding contractions. Apamin (0.1 microm) potentiated bursting activity and enhanced phasic contraction. Charybdotoxin (CTX, 50 nm) induced prolonged action potentials that generated enlarged contractions. In contrast, levcromakalim (0.1 microm) reduced the frequency of action potentials, action potential bursts and the size of the contractions. 4. Forskolin (0.1 microm), 8-bromoguanosin 3', 5' cyclic monophosphate (8Br-cGMP, 0.1 mm) and Y-26763 (10 microm) suppressed contractions without reducing the amplitude of either action potentials or Ca transients. 5. This paper confirms that action potentials and associated calcium transients are fundamental mechanisms in generating spontaneous contractions in smooth muscles of the guinea-pig bladder. However, in parallel with the excitation-contraction coupling, the sensitivity of the contractile proteins for Ca(2+) may play an important role in regulating spontaneous excitation and can be modulated by cyclic nucleotides and Rho kinase.

Figure 1

Correlation between spontaneous electrical and mechanical activity in detrusor smooth muscles of the guinea-pig bladder. A single bladder smooth muscle bundle exhibited individual action potentials (Aa) and corresponding phasic contractions (Ab). Each action potential was followed by a phasic contraction after a delay of some 0.2 s (Ac). In another preparation, bursts of action potentials (Ba) and corresponding phasic contractions were generated (Bb). Each burst was composed of some 5–20 action potentials, and was followed by a phasic contraction (Bc). Resting membrane potentials were −44 mV in (A) and −46 mV in (B).

Figure 2

Effects of CCh, apamin, CTX and CPA on the correlation of electrical and mechanical activity. In a preparation, which generated individual action potentials, CCh (0.1 μM) increased the frequency of action potentials (Aa) and contractions to cause tonic contraction (Ab). Apamin (0.1 μM) converted individual action potentials into bursts (Ba) and increased the amplitude and duration of phasic contractions (Bb). In a different preparation, which generated bursts of action potentials, CTX (50 nM) suppressed AHPs, induced action potentials that had a plateau phase (Ca) and dramatically increased the amplitude and duration of corresponding contractions (Cb). In another preparation, which generated bursts of action potentials, CPA (10 μM) suppressed AHPs, clustered the action potentials (Da), and increased the amplitude and duration of corresponding contractions (Db). Resting membrane potentials were −46 mV in (A), −44 mV in (B), −44 mV in (C) and −47 mV in (D).

Figure 3

Effects of levcromakalim on the correlation between electrical and mechanical activity. In a bladder smooth muscle preparation in which bursts of action potentials and corresponding contractions were generated (Aa, b), levcromakalim (0.1 μM) reduced the frequency of the bursts without changing either action potential shape or resting membrane potential (Ba), and reduced the frequency of the contractions (Bb). In another preparation, which generated individual action potentials, a higher concentration of levcromakalim (1 μM) hyperpolarized the membrane and prevented the generation of spontaneous action potentials (Ca). It also abolished contractions and reduced the resting tension level (Cb).

Figure 4

Effects of forskolin on the correlation between electrical and mechanical activity. In a bladder smooth muscle preparation, which exhibited individual action potentials (Aa), forskolin (0.1 μM) reduced the frequency of action potentials (Ba). It also reduced the frequency and amplitude of corresponding contractions (Bb). In another preparation, which generated bursts of action potentials, a higher concentration of forskolin (1 μM) hyperpolarized the membrane and prevented the generation of action potentials (Ca). It also abolished contractions (Cb). The resting membrane potentials were −44 mV in (A and B), and −45 mV in (C).

Figure 5

Effects of 8Br-cGMP on the correlation between electrical and mechanical activity. In a bladder smooth muscle preparation, which generated individual action potentials (Aa), 8Br-cGMP (0.1 mM) reduced the frequency of action potentials (Ba). It also reduced the frequency and amplitude of phasic contractions (Bb). In another preparation, which generated bursts of action potentials (Ca), 8Br-cGMP (0.1 mM) reduced the amplitude of corresponding contraction (Db) without affecting the bursts of action potentials (Da). The resting membrane potentials were −46 mV in (A and B) and −48 mV in (C and D).

Figure 6

The correlation between electrical and mechanical activity and intracellular calcium simultaneously recorded from guinea-pig bladder smooth muscle. In a detrusor smooth muscle preparation in which spontaneous action potentials were individually generated (Aa), each action potential and associated calcium transient (Ab) produced a corresponding contraction (Ac). On a fast time scale (Ad), the action potential (full line) was associated with a calcium transient which lasted for some 0.5 s (dotted line), and was followed by a contraction which lasted for about 1 s (dashed line). In another preparation, bursts of action potentials were generated (Bb). Each burst consisted of some 20 action potentials and was associated with a summed calcium transient (Bb) which produced a phasic contraction (Bc). The resting membrane potentials were −45 mV in (A) and −47 mV in (B).

Figure 7

Effects of forskolin, 8Br-cGMP, 2-APB and levcromakalim on the correlation between calcium transients and mechanical activity. In a bladder smooth muscle preparation, which exhibited spontaneous calcium transients (Aa) and corresponding contractions (Ac), forskolin (0.1 μM) reduced the frequency of calcium transients without changing their amplitude (Ab), and reduced the amplitude of the contractions (Ad). In the same preparation, 8Br-cGMP (0.1 mM) reduced the frequency of calcium transients without changing their amplitude (Bb), and suppressed corresponding contractions (Bd). In a different preparation, 2-APB (30 μM) increased the frequency of calcium transients without changing their amplitude (Cb), and reduced the amplitude of corresponding contractions (Cd). In another preparation, levcromakalim (0.1 μM) reduced the frequency of calcium transients without changing their amplitude (Db). It also reduced the frequency of phasic contractions but did not change their amplitude (Dd).

Figure 8

Effects of Y-27632 on electrical, calcium and mechanical activity. In a detrusor smooth muscle preparation, which exhibited individual action potentials (Aa), Y-27632 (10 μM) did not change either the amplitude or frequency of action potentials (Ba) or associated calcium transients (Bb) but it reduced the amplitude of the corresponding contractions (Bc). The resting membrane potential was −46 mV.