Prostaglandin E2 excitatory effects on guinea pig urinary bladder smooth muscle: a novel regulatory mechanism mediated by large-conductance voltage- and Ca2+-activated K+ channels

Abstract

Prostaglandin E2 (PGE2) is an essential signaling molecule involved in the regulation of detrusor smooth muscle (DSM) function. However, the underlying regulatory mechanism by which PGE2 augments DSM cell excitability and contractility is not well understood. Here, we investigated whether PGE2 inhibits the large conductance voltage- and Ca(2+)-activated K(+) (BK) channels in guinea pig DSM, thereby increasing DSM excitability and contractility. We used a multidisciplinary experimental approach including amphotericin-B perforated patch-clamp electrophysiology and live-cell Ca(2+) imaging in native freshly-isolated DSM cells, isometric tension recordings of intact DSM strips, and pharmacological tools to investigate BK channel regulation by PGE2 in guinea pig DSM. PGE2 increased the spontaneous phasic contractions of isolated DSM strips in a concentration-dependent manner (10 nM-10 µM). BK channel inhibition with paxilline (1 µM) attenuated the PGE2-induced DSM phasic contractions, suggesting that BK channels are involved in the mechanism of PGE2-induced DSM contractions. PGE2 (10 µM) increased the intracellular Ca(2+) levels in freshly-isolated DSM cells. PGE2 (10 µM) also caused an inhibition of the amplitude and frequency of spontaneous transient BK currents in DSM cells. Moreover, PGE2 (10 µM) did not affect the amplitude of whole cell steady-state BK currents in DSM cells. Our findings provide strong experimental evidence that PGE2 leads to an inhibition of the spontaneous transient BK currents, elevation of intracellular Ca(2+) levels in freshly-isolated DSM cells, and augmentation of DSM phasic contractions. Thus, we have revealed a novel mechanism that BK channels mediate PGE2-induced contractions in guinea pig DSM.

Keywords: Ca(2+) imaging; Detrusor; Patch-clamp; Prostaglandin E(2).

Fig. 1. PGE₂ significantly increases the spontaneous phasic contractions of guinea pig isolated DSM strips. A and B)

Representative original recordings showing the contractile effects of PGE₂ (10 nM-10 μM) on spontaneous phasic contractions of isolated DSM strips in the absence (A) or presence (B) of paxilline (1 μM). C and D) Cumulative concentration-response curves for PGE₂ on spontaneous phasic contraction amplitude and muscle force integral of DSM strips in the presence or absence of 1 μM paxilline (n=8, N=5; ^#P<0.05, ^##P<0.01, and ^###P<0.001 PGE₂ vs. control, *P<0.05 and **P<0.01 vs. paxilline).

Fig. 2. PGE₂ increases the intracellular Ca²⁺ levels in native freshly-isolated guinea pig DSM cells. A)

A representative trace of fura 2 fluorescence ratio illustrating that PGE₂ (10 μM) increases the intracellular Ca²⁺ levels in a DSM cell. B) Summary data depicting the DSM cell intracellular Ca²⁺ levels in the presence or absence of PGE₂ (10 μM) (n=6, N=6; * P<0.05).

Fig. 3. PGE₂ inhibits TBKCs in freshly-isolated guinea pig DSM cells. A)

A representative recording illustrating the PGE₂ (10 μM) inhibitory effects on the TBKC amplitude and frequency in a freshly-isolated DSM cell. B) Summary data illustrating the inhibitory effect of PGE₂ (10 μM) on the amplitude and frequency of TBKCs (n=12, N=7; *P<0.05). The average amplitude and frequency of TBKCs before PGE₂ application (control) were taken to be 100% and data were normalized to controls.

Fig. 4. PGE₂ does not affect the steady-state whole cell BK currents in freshly-isolated guinea pig DSM cells. A)

Representative voltage-clamp recordings illustrating that PGE₂ (10 μM) does not affect the steady-state BK currents at depolarization voltages from −40 mV to +80 mV. B) Current-voltage relationship curves depict the lack of PGE₂ effect on the steady-state whole cell BK currents in DSM cells (n=6, N=6; P>0.05).