Constitutive PKA activity is essential for maintaining the excitability and contractility in guinea pig urinary bladder smooth muscle: role of the BK channel

Abstract

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca(2+) sparks but increased global Ca(2+) levels and the magnitude of Ca(2+) oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca(2+) signals generated by intracellular Ca(2+) stores and cytosolic Ca(2+) levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM.

Keywords: Ca2+ sparks; H-89; PKI 14–22; large-conductance voltage- and Ca2+-activated K+ channels; protein kinase A.

Fig. 1.

Pharmacological inhibition of PKA with N-{2-[3-(4-bromophenyl)-2-propenylamino]ethyl}-5-isoquinolinesulfonamide dihydrochloride (H-89) or protein kinase A inhibitor PKI 14–22 reduced the spontaneous transient large-conductance voltage- and Ca²⁺-activated K⁺ channel (BK) channel currents (TBKCs) in freshly-isolated urinary bladder smooth muscle (UBSM) cells. A and B: original voltage clamp recordings from single UBSM cells illustrating that H-89 (10 μM) or PKI 14–22 (5 μM) inhibited TBKCs. A portion of the recording under each condition is shown on an expanded time scale. C and D: summary data illustrating that H-89 (10 μM; n = 6, N = 6) and PKI 14–22 (5 μM; n = 6, N = 5) significantly decreased the frequency and amplitude of TBKCs (n = no. of cells, and N = no. of guinea pigs). Freq, frequency; Amp, amplitude. *P < 0.05.

Fig. 2.

Pharmacological inhibition of PKG with DT-2 did not have any effect on the TBKCs in freshly isolated UBSM cells. A: an original voltage clamp recording illustrating that DT-2 (1 μM) did not affect TBKCs and that the subsequent PKA inhibition with H-89 completely inhibited TBKCs in a UBSM cell. A portion of the recording under each condition is shown on an expanded time scale. B: summary data illustrating that DT-2 (1 μM) did not have a significant effect on the frequency (Freq) and amplitude (Amp) of TBKCs (n = 6, N = 5). P > 0.05.

Fig. 3.

Pharmacological inhibition of PKA with H-89 suppressed the basal level of Ca²⁺ sparks in freshly isolated UBSM cells. A: an image of a freshly isolated UBSM cell loaded with fluo 4-AM. The white line passing through the active site a is the laser beam-scanning pathway (1-pixel width). B: the 3-dimensional view of the recordings illustrate the Ca²⁺ sparks in the absence and presence of H-89 (10 μM). The color scale indicates the relative fluorescence intensity F/F₀. C: summary data illustrating that H-89 (10 μM) decreased the Ca²⁺ spark frequency (Freq) and amplitude (Amp) in UBSM cells (n = 8, N = 6) *P < 0.05.

Fig. 4.

Pharmacological inhibition of PKA with H-89 increased the global cellular Ca²⁺ levels and the magnitude of Ca²⁺ oscillations in freshly isolated UBSM cells. A: an original recording illustrating that H-89 increased the global Ca²⁺ levels in a freshly isolated UBSM cell. B: summary data show that the inhibition of PKA significantly increased the global intracellular Ca²⁺ levels in UBSM cells (n = 8, N = 4). C: the overlay of 100 s of recordings under control conditions and after H-89 treatment shows that H-89 increased the oscillation of the cytosolic Ca²⁺ in UBSM cells. D: summary data show that the inhibition of PKA significantly increased the magnitude of Ca²⁺ oscillations, which is measured as the standard deviation (SD) of the F₃₄₀/F₃₈₀ ratio during the last 5 min of recording in the absence and presence of H-89 (10 μM; n = 8, N = 4). *P < 0.05. Con, control.

Fig. 5.

Pharmacological inhibition of PKA with H-89 abolished TBKCs and prevented caffeine-induced outward current in freshly isolated UBSM cells. A: an original voltage clamp recording illustrating that caffeine (10 mM), a ryanodine receptor (RyR) opener, transiently increased the outward currents and then abolished TBKCs in a single UBSM cell (n = 9, N = 6). B: an original recording illustrating that PKA inhibition with H-89 (10 μM) abolished TBKCs and that the subsequent activation of RyR with 10 mM caffeine did not induce any outward currents in single UBSM cells (n = 5, N = 5).

Fig. 6.

Pharmacological inhibition of PKA with H-89 abolished the spontaneous transient hyperpolarizations and depolarized UBSM cell resting membrane potential. A: an original current clamp recording illustrating that H-89 (10 μM) inhibited the spontaneous transient hyperpolarizations in an isolated UBSM cell. A portion of the recording under each condition is shown on an expanded time scale. B: summary data illustrating that H-89 (10 μM) depolarized the membrane potential of UBSM cells (n = 11, N = 8). *P < 0.05.

Fig. 7.

Pharmacological inhibition of PKA with H-89 or KT-5720 significantly increased spontaneous phasic contractions of isolated UBSM strips. A and B: representative recordings of UBSM strips illustrating that pharmacological PKA inhibition increases UBSM spontaneous phasic contractions. C and D: summary data illustrating the effects of PKA inhibition with H-89 (10 μM; n = 14, N = 10) or KT-5720 (3 μM; n = 8, N = 5) on the amplitude (Amp), muscle force integral (Force), duration (Dur), frequency (Freq), and tone of UBSM spontaneous phasic contractions. *P < 0.05.

Fig. 8.

A diagram depicting the signaling pathway by which the constitutive PKA activity regulates the spontaneous TBKCs and thus UBSM excitability and contractility. AC, adenylyl cyclase; Ca_V, L-type voltage-gated Ca²⁺ channel; pPLB, phosphorylated phospholamban; SR, sarcoplasmic reticulum.