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.

BK channel regulation by phosphodiesterase type 1: a novel signaling pathway controlling human detrusor smooth muscle function

“…In the presence of 8MM-IBMX, H-89 (10 M) reduced the mean values for TBKC frequency and amplitude to 11.0 Ϯ 5.7 and 45.6 Ϯ 22.9% of the control, respectively (n ϭ 3, N ϭ 3). Reduction of TBKC frequency and amplitude to levels below the control implies constitutive PKA activity as we recently reported (27).…”

“…The activation of BK channels hyperpolarizes the human DSM cell membrane potential (13,28). The pharmacological inhibition of constitutive PKA activity abolishes TBKCs and depolarizes DSM cell membrane potential (27) (Figs. 1 and 3).…”

“…6) (6). A decrease in basal PKA activity significantly attenuates BK channels in DSM cells (19,27). However, this concept needs further investigation as it is particularly important to also study the enzymatic kinetics of PDE1 regarding its regulation by mAChRs in human DSM.…”

“…BK channel activity is regulated by localized intracellular Ca 2ϩ release events from the sarcoplasmic reticulum (SR) ryanodine receptors (RyRs), known as "Ca 2ϩ sparks." Ca 2ϩ sparks transiently activate BK channels generating transient BK currents (TBKCs) (8,12,16,27,28). TBKCs correspond to transient hyperpolarizations in DSM cells (19).…”

Attenuated BK channel function promotes overactive bladder in a rat model of obesity

“…Enhanced BK channel activity induces spontaneous transient outward currents (STOCs), which hyperpolarize the membrane and reduce the influx of Ca 2+ by attenuating the activity of voltage-dependent Ca 2+ channels. These effects ultimately relax the DSM [20–22].…”