Actions of cAMP on calcium sensitization in human detrusor smooth muscle contraction.

OBJECTIVES: To clarify the effect of cAMP on the Ca(2+) -sensitized smooth muscle contraction in human detrusor, as well as the role of novel exchange protein directly activated by cAMP (Epac) in cAMP-mediated relaxation. MATERIALS AND METHODS: All experimental protocols to record isometric tension force were performed using α-toxin-permeabilized human detrusor smooth muscle strips. The mechanisms of cAMP-mediated suppression of Ca(2+) sensitization activated by 10 µm carbachol (CCh) and 100 µm GTP were studied using a selective rho kinase (ROK) inhibitor, Y-27632, and a selective protein kinase C (PKC) inhibitor, GF-109203X. The relaxation mechanisms were further probed using a selective protein kinase A (PKA) activator, 6-Bnz-cAMP and a selective Epac activator, 8-pCPT-2'-O-Me-cAMP. RESULTS: We observed that CCh-induced Ca(2+) sensitization was inhibited by cAMP in a concentration-dependent manner. GF-109203X (10 µm) but not Y-27632 (10 µm) significantly enhanced the relaxation effect induced by cAMP (100 µm). 6-Bnz-cAMP (100 µm) predominantly decreased the tension force in comparison with 8-pCPT-2'-O-Me-cAMP (100 µm). CONCLUSIONS: We showed that cAMP predominantly inhibited the ROK pathway but not the PKC pathway. The PKA-dependent pathway is dominant, while Epac plays a minor role in human detrusor smooth muscle Ca(2+) sensitization.

Novel effect of 2-aminoethoxydiphenylborate through inhibition of calcium sensitization induced by Rho kinase activation in human detrusor smooth muscle.

Since the introduction of 2-aminoethoxydiphenylborate (2-APB) as a membrane permeable modulator of inositol (1,4,5)-trisphosphate receptors, subsequent studies have revealed additional actions of this chemical on multiple Ca(2+)-permeable ionic channels in the plasma membrane. However, no reports have yet examined 2-APB as a modulator targeting contractile machinery in smooth muscle, independent of Ca(2+) mobilization, namely Ca(2+) sensitization. Here, we assessed whether or not 2-APB affects intracellular signaling pathways of Ca(2+) sensitization for contraction using α-toxin permeabilized human detrusor smooth muscle. Although contractions were induced by application of Ca(2+)-containing bath solutions, 2-APB had little effect on contractions induced by 1 µM Ca(2+) alone but significantly reversed the carbachol-induced augmentation of Ca(2+)-induced contraction in the presence of guanosine triphosphate (carbachol-induced Ca(2+) sensitization). The rho kinase inhibitor Y-27632 and protein kinase C inhibitor GF-109203X also reversed the carbachol-mediated Ca(2+) sensitization. Additional application of 2-APB caused a small but significant further attenuation of the contraction in the presence of GF-109203X but not in the presence of Y-27632. Like carbachol, the rho kinase activator; sphingosylphosphorylcholine, protein kinase C activator; phorbol 12,13 dibutyrate, and myosin light chain phosphatase inhibitor; calyculin-A all induced Ca(2+) sensitization. However, the inhibitory activity of 2-APB was limited with sphingosylphosphorylcholine-induced Ca(2+) sensitization. This study revealed a novel inhibitory effect of 2-APB on smooth muscle contractility through inhibition of the rho kinase pathway.

Endogenous cardiac troponin T modulates Ca(2+)-mediated smooth muscle contraction.

Mechanisms linked to actin filaments have long been thought to cooperate in smooth muscle contraction, although key molecules were unclear. We show evidence that cardiac troponin T (cTnT) substantially contributes to Ca(2+)-mediated contraction in a physiological range of cytosolic Ca(2+) concentration ([Ca(2+)](i)). cTnT was detected in various smooth muscles of the aorta, trachea, gut and urinary bladder, including in humans. Also, cTnT was distributed along with tropomyosin in smooth muscle cells, suggesting that these proteins are ready to cause smooth muscle contraction. In chemically permeabilised smooth muscle of cTnT(+/-) mice in which cTnT reduced to ~50%, the Ca(2+)-force relationship was shifted toward greater [Ca(2+)](i), indicating a sizeable contribution of cTnT to smooth muscle contraction at [Ca(2+)](i) < 1 µM. Furthermore, addition of supplemental TnI and TnC reconstructed a troponin system to enhance contraction. The results indicated that a Tn/Tn-like system on actin-filaments cooperates together with the thick-filament pathway.

Functional role of muscarinic receptor subtypes in calcium sensitization and their contribution to rho-kinase and protein kinase C pathways in contraction of human detrusor smooth muscle.

OBJECTIVE: We investigated the role of muscarinic receptor subtypes in calcium sensitization and their contribution to rho-kinase (ROK) and protein kinase C (PKC) pathways in carbachol (CCh)-induced contraction of human detrusor smooth muscle (DSM). MATERIALS AND METHODS: α-toxin-permeabilized human DSM strips were prepared and mounted horizontally to record isometric force. The roles of M(2) and M(3) muscarinic receptors in Ca(2+) sensitization were studied using selective antagonists of M(2) (AF-DX116) and M(3) (4-DAMP) receptor subtypes. The effects of a selective inhibitor of ROK, Y-27632, and a selective inhibitor of PKC, bisindolylmaleimide I (GF-109203X), were also studied on contraction induced by 10 µM CCh with 100 µM guanosine triphosphate at a fixed 1 µM [Ca(2+)](i) after preincubation with 1 µM AF-DX116 or 1 µM 4-DAMP. RESULTS: Carbachol-induced Ca(2+) sensitization was predominantly inhibited by 4-DAMP compared with AF-DX116. Four-DAMP equivalently inhibited the relaxation effect of 5 µM GF-109203X as well as that of 5 µM Y-27632 on CCh-induced Ca(2+) sensitization. AF-DX116 reduced the relaxation effect of Y-27632 to a greater degree than GF-109203X. CONCLUSION: The results of the present study have demonstrated the predominant role of M(3) receptor subtype in Ca(2+) sensitization and the relative contribution to ROK and PKC pathways. Our study also shows that the ROK pathway is dominant compared with the PKC pathway after M(2) receptor activation, which in turn is inferior, but not negligible, in producing Ca(2+) sensitization.

Obstruction enhances rho-kinase pathway and diminishes protein kinase C pathway in carbachol-induced calcium sensitization in contraction of α-toxin permeabilized guinea pig detrusor smooth muscle.

AIMS: We investigated the relative important role of rho kinase (ROK) and protein kinase C (PKC) pathways in carbachol (CCh)-induced Ca(2+) sensitization in α-toxin permeabilized Guinea pig detrusor smooth muscle (DSM) following bladder outlet obstruction (BOO). METHODS: Bladder outlet obstruction was created by placement of a silver jeweler's jump rings loosely round the urethro-vesical junction of Guinea pigs. Sham operated Guinea pig underwent a similar protocol without application of the ring and served as control. α-Toxin permeabilized DSM strips from control Guinea pigs and those subjected to 6-8 weeks of BOO were mounted horizontally for isometric force recording in 100 µl relaxing solution on perspex block. The effect of ROK inhibitor (Y-27632) and PKC inhibitor (GF-109203X) on CCh-induced Ca(2+) sensitization was studied during sustained contraction. Permeabilized DSM strips were also stimulated by cumulative increase of Ca(2+) concentration compared to that in control in the presence and in the absence of sensitization-induced PKC activator, phorbol 12,13-dibutyrate. RESULTS: Ca(2+) sensitization-induced by CCh was greater in BOO compared to controls. This muscarinic agonist-induced Ca(2+) sensitization was inhibited by Y-27632 or GF-109203X. The inhibitory effect of Y-27632 (5 µM) was greater while the inhibitory effect of GF-109203X (5 µM) was smaller in BOO compared to that in controls. Phorbol 12,13-dibutyrate (1 µM) markedly increased Ca(2+) sensitivity in controls but not in BOO. CONCLUSIONS: Our findings provide the first evidence that BOO enhances the ROK pathway and diminishes the PKC pathway in CCh-induced Ca(2+) sensitization in contraction of permeabilized Guinea pig DSM and suggest that inhibitors of ROK might potentially relieve bladder dysfunction related to BOO.

Diphosphate regulation of adenosine triphosphate sensitive potassium channel in human bladder smooth muscle cells.

PURPOSE: To clarify the properties of adenosine triphosphate sensitive K+ channel in human detrusor smooth muscle we examined the effect of the representative nicotinic acid derivatives ß-nicotinamide adenine dinucleotide, cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate (Sigma-Aldrich®) on human detrusor adenosine triphosphate sensitive K+ channels. MATERIALS AND METHODS: Patch clamp procedures were done in human detrusor cells. Reverse transcriptase and real-time polymerase chain reaction were performed to clarify the subunit components of adenosine triphosphate sensitive K+ channels. RESULTS: The K+ channel opener levcromakalim induced a long lasting outward current that was inhibited by glibenclamide (Sigma-Aldrich) under the whole cell configuration. The single channel study revealed that the unitary conductance of the adenosine triphosphate sensitive K+ channel in the human detrusor was 11 pS and nucleotide diphosphates increased its open probability. Applying ß-nicotinamide adenine dinucleotide also activated the adenosine triphosphate sensitive K+ channel but applying cyclic adenosine diphosphate ribose or nicotinic acid adenine dinucleotide phosphate had little effect on channel activation. Molecular studies indicated that Kir6.1 and SUR2B were the predominant components of the adenosine triphosphate sensitive K+ channel in the human detrusor. CONCLUSIONS: To our knowledge we report the first single channel study of the adenosine triphosphate sensitive K+ channel in the human detrusor. The properties of this channel, ie unitary conductance, adenosine triphosphate sensitivity and diphosphate activation, were consistent with those of other smooth muscle organs. ß-Nicotinamide adenine dinucleotide has the potency to activate adenosine triphosphate sensitive K+ channels in the human detrusor. This channel likely has some role during ischemic conditions as well as physiological muscle motion leading to the activation of cell metabolism.

Voiding dynamics in women with stress urinary incontinence and high-stage cystocele.

OBJECTIVES: The aim of this study was to identify the urodynamic features of women with stress urinary incontinence (SUI) or with high-stage (stage 3 or greater) cystocele (HSC) as compared with symptom-free women. METHODS: Fifty-six neurologically intact women with SUI and 47 women with HSC but without SUI were prospectively evaluated. All patients underwent full urodynamics, in addition to basic clinical evaluations. The urodynamic parameters of SUI and HSC were compared to the ones obtained from 78 urologically symptom-free normal women over the same period. RESULTS: Patients with HSC, after correction of cystocele using a temporary vaginal pessary, had consistently lower maximum urinary flow rate with a lower detrusor pressure during micturition than the controls or those with SUI. On the other hand, patients with SUI had an equivalent to higher maximum urinary flow rate, normal detrusor contraction strength with a lower detrusor pressure during micturition than the controls. Both maximum Watts factor and bladder contractility index were significantly lower in the whole HSC cohort in comparison to the controls and patients with SUI. The urodynamic characteristics observed among the three groups were all maintained even after adjusting for age. CONCLUSIONS: Women with SUI demonstrate voiding with low-pressure, normal contraction strength with an equivalent to high urinary flow rate. Women with HSC demonstrate voiding with low pressures with weak contraction strengths and low urinary flow rates, suggesting a higher prevalence of detrusor underactivity. Chronically decreased or increased urethral resistance might alter voiding dynamics and performance.

The profiles and patterns of detrusor overactivity and their association with overactive bladder symptoms in men with benign prostatic enlargement associated with detrusor overactivity.

AIMS: To investigate the association of both the urodynamic profiles and patterns of detrusor overactivity (DO) with the severity of the symptoms related to overactive bladder (OAB) in men with benign prostatic enlargement (BPE) associated with DO. METHODS: A total of 777 men with symptomatic BPE who had undergone transurethral resection of the prostate (TURP) were retrospectively analyzed. All 231 cases of the 317 cases associated with DO were enrolled into the final analysis. The severity of the preoperative symptoms related to OAB were graded based on the International Prostate Symptoms Score (IPSS). The correlation between DO profiles and the severity of OAB related symptoms were analysed using Spearman's correlation test while the association of the DO patterns with the severity of symptoms related OAB were analysed using the Mann Whitney test. Values of P less than 0.05 were considered to be statistically significant. RESULTS: A significant negative correlation was found between the severity of nocturia symptoms and bladder filling volume at the beginning of the first DO. The severity of nocturia symptoms, the amplitude of DO pressure and DO(time) were significantly increased with increasing age. The severity of nocturia symptom was significantly higher in single DO in comparison to that in multiple DO. CONCLUSIONS: The significant association of the severity of nocturia symptoms with bladder filling volume at the beginning of the first DO, age and the number of DO pattern was established. The amplitude and duration of DO may affect the severity of the symptoms.