External Validation of a Prognostic Model Predicting Metastatic Castration-Resistant Prostate Cancer Survival in Patients Receiving Post-Docetaxel Second-Line Chemotherapy.

Introduction: The Halabi model predicts the overall survival (OS) of patients with metastatic castration-resistant prostate cancer (mCRPC) treated with second-line therapy after docetaxel. We aimed to validate this model externally with an independent cohort, outside the setting of a clinical trial. Methods: In a multi-institutional study, we included 66 patients treated with cabazitaxel after docetaxel for mCRPC. Patients were stratified according to the two- and three-risk groups of the Halabi nomogram. Kaplan-Meier and Cox proportional hazard analyses were performed to estimate survival and hazard ratios (HRs). The model performance was assessed using receiver operating characteristic curves, and the associated c-index (area under the curve [AUC]). Results: The median OS in the two-risk groups was 5.06 months in the high-risk group (n=22) and 12.9 months in the low-risk group (n=44, p<0.001). High-risk patients had an HR of 9.50 (95% confidence interval (CI) 4.12-21.6, p<0.001) compared to low-risk patients. For the three-risk groups, the median OS was 6.44 months in the high-risk group (n=15), 5.75 months in the intermediate-risk group (n=11), and 13.7 months in the low-risk group (n=40, p=0.84). Compared to low-risk patients, intermediate-risk patients had an HR of 7.49 (95% CI 3.08-20.4, p<0.001), and high-risk patients had an HR of 8.48 (95% CI, 3.39-21.7, p<0.001). The AUC was 0.72 (95% CI 0.64-0.76) for the two-risk stratification. When comparing different risks, the AUCs were 0.48 (high vs intermediate), 0.66 (high vs low), and 0.65 (intermediate vs low). Conclusions: The two-risk stratification version but not the three-risk group analysis confirmed the ability of the model to predict survival. These results support the value of the Halabi nomogram in men receiving post-docetaxel second-line chemotherapy for mCRPC.

Efficacy and safety of cabazitaxel therapy in elderly (≥75 years) patients with castration-resistant prostate cancer: A multiinstitutional study.

BACKGROUND: There is little data on the outcome of cabazitaxel (CBZ) treatment of elderly patients with castration-resistant prostate cancer (CRPC). This study assessed the efficacy and safety of CBZ chemotherapy in patients with CRPC aged 75 years or older in a multiinstitutional study. METHODS: We retrospectively reviewed the 74 patients with CRPC treated with CBZ enrolled in 10 institutions. Clinicopathological backgrounds, prognosis including prostate-specific antigen decline, time to treatment failure, progression-free survival, overall survival, and safety profiles were compared between younger (<75 years) and elder (≥75 years) patients. RESULTS: In total, 74 patients were enrolled; 50 patients were younger than 75 years and 24 were ≥75 years. Clinicopathological characteristics were comparable between younger and elder patients, with the exception of serum albumin values at the time of CBZ treatment. The median prostate-specific antigen decline in younger and elder men was -8.8% and -32.3% from baseline, respectively. The median time to treatment failure, progression-free survival, and overall survival for younger and elder men were 0.24 and 0.33 years, 0.23 and 0.43 years, and 0.69 and 1.17 years, respectively. In addition, safety profiles were comparable between younger and elder patients. CONCLUSIONS: This multiinstitutional study suggests that patients with CRPC aged 75 years or older eligible for CBZ treatment can be treated safely and with noninferior efficacy compared with those younger than 75 years.

Prognostic significance of lactate dehydrogenase in cabazitaxel chemotherapy for castration-resistant prostate cancer: a multi-institutional study.

This multi-institutional study aimed to identify prognostic factors for cabazitaxel treatment of castration-resistant prostate cancer (CRPC). This study included 74 Japanese patients with CRPC who were treated with cabazitaxel between 2014 and 2017. Associations between clinicopathological factors including serum markers and progression-free survival (PFS) and overall survival (OS) were investigated. On multivariate analysis, high Gleason score [≥9 vs. ≤7; hazard ratio (HR), 95% confidence interval (CI): 2.00 (1.01-4.34); P = 0.047], presence of pain [HR, 95% CI: 2.02 (1.14-3.58); P = 0.016], and lactate dehydrogenase (LDH) level [HR, 95% CI: 47.31 (3.79-577.49); P = 0.0019] were significantly associated with PFS. Similarly, number of docetaxel cycles [HR, 95% CI: 0.050 (0.0037-0.45); P = 0.0057], performance status [≥2 vs. 0; HR, 95% CI: 5.07 (1.57-16.24); P < 0.0001], and LDH level [HR, 95% CI: 2946 (50-420994); P = 0.0001] were significantly associated with OS. This study showed that LDH level is robustly prognostic for both PFS and OS in cabazitaxel chemotherapy for CRPC.

Prognostic Impact of Prior Androgen Receptor Axis-targeting Agents in Cabazitaxel Chemotherapy After Docetaxel.

BACKGROUND/AIM: The novel taxane cabazitaxel has been shown to exert excellent anticancer effects after androgen receptor axis-targeting (ARAT) agents in clinical data, but not in in vitro data. We investigated the clinical outcome of cabazitaxel chemotherapy after docetaxel according to use of ARAT agents. PATIENTS AND METHODS: Prostate specific antigen (PSA) response, progression-free survival, and overall survival were compared between cases with and without prior use of ARAT agents in 74 Japanese patients with metastatic castration-resistant prostate cancer treated with cabazitaxel chemotherapy. RESULTS: Background characteristics were comparable between patients with and without prior use of ARAT agents. PSA response, progression-free survival, and overall survival in cabazitaxel chemotherapy were comparable between patients with and without prior use of ARAT agents. CONCLUSION: No detrimental effects of prior ARAT agents on clinical outcome were observed for cabazitaxel chemotherapy in the post-docetaxel setting, suggesting that cabazitaxel can be expected to remain active even after ARAT agent therapy.

Therapeutic Outcome of >10 Cycles of Cabazitaxel for Castration-resistant Prostate Cancer: A Multi-institutional Study.

BACKGROUND/AIM: Cabazitaxel use has usually been limited to up to 10 cycles in most countries according to the protocol in the TROPIC trial. Therefore, clinical data on cabazitaxel use beyond 10 cycles is limited. The aim of this study was to report the therapeutic outcome of cabazitaxel chemotherapy administered for >10 cycles. PATIENTS AND METHODS: This study included 74 Japanese patients with prostate cancer between 2014 and 2017. Patients background, and treatment outcomes including PSA decline, progression-free survival, treatment-failure-free survival, overall survival, and adverse events were investigated, comparing patients treated with ≤10 and >10 cycles. RESULTS: Patients characteristics were favorable as indicated by the higher number of cycles of prior docetaxel chemotherapy, absence of pain, and absence of bony and visceral metastases among men who received >10 cycles of cabazitaxel. PSA response, progression-free survival, treatment-failure-free survival and overall survival were better among patients treated with >10 cycles of cabazitaxel compared to those treated with ≤10 cycles. The incidence of severe adverse events was similar between the two groups. CONCLUSION: Taken together, this study suggested that continuous chemotherapy with cabazitaxel beyond 10 cycles may be beneficial.

Efficacy and safety of 4-weekly cabazitaxel for castration-resistant prostate cancer: a multi-institutional study.

OBJECTIVE: This study aimed to reveal the efficacy and safety profiles of 4-weekly cabazitaxel in patients with castration-resistant prostate cancer (CRPC). METHODS: The study included 62 Japanese patients who were treated for CRPC with ≥ 2 courses of cabazitaxel between 2014 and 2017. The oncological outcomes and adverse events were compared between 16 (25.8%) and 46 (74.2%) men who were treated with standard 3-weekly and alternative 4-weekly regimens, respectively. RESULTS: The prostate-specific antigen (PSA) response was comparable between the 3-weekly and 4-weekly regimens (median [interquartile range]: - 9.9% [- 64.5 to 13.0%] and - 30.7% [- 52.8 to 10.9%], P = 0.89), respectively. For patients on the 4-weekly regimen, the risks of progression (hazard ratio [HR], 95% confidence interval [CI] 1.27, 0.71-2.43, P = 0.44), treatment failure (HR, 95% CI 0.84, 0.48-1.55, P = 0.57) and any-cause mortality (HR, 95% CI 1.09, 0.58-2.17, P = 0.79) were comparable to those for patients on the 3-weekly regimen. The incidences of severe adverse events were also similar between the 3-weekly and 4-weekly regimens. CONCLUSIONS: 3-weekly and 4-weekly regimens of cabazitaxel showed similar efficacy and safety profiles in a real-world clinical setting. These data suggest that a 4-weekly regimen may be acceptable for selected patients.

Efficacy and safety of cabazitaxel for castration-resistant prostate cancer in patients with > 10 cycles of docetaxel chemotherapy: a multi-institutional study.

This multi-institutional study aimed to investigate the efficacy and safety profiles of cabazitaxel after prior docetaxel chemotherapy in patients with castration-resistant prostate cancer (CRPC). This study included 63 Japanese patients with CRPC who were treated with cabazitaxel from 2014 to 2017. The oncological outcomes and adverse events (AEs) were documented, and prognostic factors for oncological outcomes and predictive factors for AEs were analysed. PSA decline was observed in 68.3% of patients, including 25.4% who achieved a ≥ 50% decline. The median progression-free survival, treatment failure-free survival, and overall survival were 4.3, 4.1, and 9.0 months, respectively. More cycles of prior docetaxel therapy was identified as common favourable prognostic factors for progression-free survival, treatment failure-free survival, and overall survival. Severe neutropenia, febrile neutropenia, and severe non-haematological AEs were observed in 73.0%, 33.3%, and 23.8% of patients, respectively. However, > 10 cycles of docetaxel was not associated with increased incidence of AEs. In conclusion, cabazitaxel chemotherapy was still active in Japanese CRPC patients treated with > 10 cycles of docetaxel chemotherapy, with an acceptable risk of AE burden. Treatment with cabazitaxel after > 10 cycles of docetaxel may be an appropriate option when it can be administered.

Differential contribution of Kv4-containing channels to A-type, voltage-gated potassium currents in somatic and visceral dorsal root ganglion neurons.

Little is known about electrophysiological differences of A-type transient K(+) (KA) currents in nociceptive afferent neurons that innervate somatic and visceral tissues. Staining with isolectin B4 (IB4)-FITC classifies L6-S1 dorsal root ganglion (DRG) neurons into three populations with distinct staining intensities: negative to weak, moderate, and intense fluorescence signals. All IB4 intensely stained cells are negative for a fluorescent dye, Fast Blue (FB), injected into the bladder wall, whereas a fraction of somatic neurons labeled by FB, injected to the external urethral dermis, is intensely stained with IB4. In whole-cell, patch-clamp recordings, phrixotoxin 2 (PaTx2), a voltage-gated K(+) (Kv)4 channel blocker, exhibits voltage-independent inhibition of the KA current in IB4 intensely stained cells but not the one in bladder-innervating cells. The toxin also shows voltage-independent inhibition of heterologously expressed Kv4.1 current, whereas its inhibition of Kv4.2 and Kv4.3 currents is voltage dependent. The swapping of four amino acids at the carboxyl portion of the S3 region between Kv4.1 and Kv4.2 transfers this characteristic. RT-PCRs detected Kv4.1 and the long isoform of Kv4.3 mRNAs without significant Kv4.2 mRNA in L6-S1 DRGs. Kv4.1 and Kv4.3 mRNA levels were higher in laser-captured, IB4-stained neurons than in bladder afferent neurons. These results indicate that PaTx2 acts differently on channels in the Kv4 family and that Kv4.1 and possibly Kv4.3 subunits functionally participate in the formation of KA channels in a subpopulation of somatic C-fiber neurons but not in visceral C-fiber neurons innervating the bladder.

Hyperexcitability of bladder afferent neurons associated with reduction of Kv1.4 α-subunit in rats with spinal cord injury.

PURPOSE: To clarify the functional and molecular mechanisms inducing hyperexcitability of C-fiber bladder afferent pathways after spinal cord injury we examined changes in the electrophysiological properties of bladder afferent neurons, focusing especially on voltage-gated K channels. MATERIALS AND METHODS: Freshly dissociated L6-S1 dorsal root ganglion neurons were prepared from female spinal intact and spinal transected (T9-T10 transection) Sprague Dawley® rats. Whole cell patch clamp recordings were performed on individual bladder afferent neurons. Kv1.2 and Kv1.4 α-subunit expression levels were also evaluated by immunohistochemical and real-time polymerase chain reaction methods. RESULTS: Capsaicin sensitive bladder afferent neurons from spinal transected rats showed increased cell excitability, as evidenced by lower spike activation thresholds and a tonic firing pattern. The peak density of transient A-type K+ currents in capsaicin sensitive bladder afferent neurons from spinal transected rats was significantly less than that from spinal intact rats. Also, the KA current inactivation curve was displaced to more hyperpolarized levels after spinal transection. The protein and mRNA expression of Kv1.4 α-subunits, which can form transient A-type K+ channels, was decreased in bladder afferent neurons after spinal transection. CONCLUSIONS: Results indicate that the excitability of capsaicin sensitive C-fiber bladder afferent neurons is increased in association with reductions in transient A-type K+ current density and Kv1.4 α-subunit expression in injured rats. Thus, the Kv1.4 α-subunit could be a molecular target for treating overactive bladder due to neurogenic detrusor overactivity.

Differential effects of botulinum neurotoxin A on bladder contractile responses to activation of efferent nerves, smooth muscles and afferent nerves in rats.

PURPOSE: To determine the mechanisms of botulinum neurotoxin A (Metabiologics, Madison, Wisconsin) induced inhibition of bladder activity we examined the effect of botulinum neurotoxin A on detrusor contractile responses to the activation of L-type voltage-gated Ca(2+) channels, and efferent and afferent nerve terminals in the rat bladder. MATERIALS AND METHODS: Rat bladder strips were incubated for 3 hours with different concentrations of botulinum neurotoxin A (0.3 to 100 nM). We examined the effect of botulinum neurotoxin A on detrusor contractility in response to activation of L-type voltage-gated Ca(2+) channels, and efferent and afferent nerve terminals induced by 70 mM KCl, electrical field stimulation and 1 µM capsaicin, respectively. RESULTS: Botulinum neurotoxin A inhibited electrical field stimulation induced contractions at a concentration of 10 nM or higher. The maximal inhibition at 100 nM was 70% compared to that of control strips. KCl induced contractions, which were sensitive to nifedipine, were significantly inhibited by incubation with botulinum neurotoxin A at a concentration of 3 nM or higher. Maximal inhibition at 100 nM was 30% compared to that of control strips. Capsaicin induced contractions were not inhibited by 3-hour incubation but they were significantly inhibited by overnight incubation with 100 nM botulinum neurotoxin A (30% compared to control strips). Carbachol induced contractions were not altered by incubation with botulinum neurotoxin A. CONCLUSIONS: The order of inhibitory potency of botulinum neurotoxin A was efferent nerve terminals >L-type voltage-gated Ca(2+) channels >afferent nerve terminals. Since the inhibitory effects on L-type voltage-gated Ca(2+) channels and efferent nerve terminals were observed at similar botulinum neurotoxin A concentrations, the inhibitory effect of botulinum neurotoxin A on L-type voltage-gated Ca(2+) channels may have an important role in regulating and stabilizing bladder activity.

Distinct cellular distributions of Kv4 pore-forming and auxiliary subunits in rat dorsal root ganglion neurons.

AIMS: Dorsal root ganglia contain heterogeneous populations of primary afferent neurons that transmit various sensory stimuli. This functional diversity may be correlated with differential expression of voltage-gated K(+) (Kv) channels. Here, we examine cellular distributions of Kv4 pore-forming and ancillary subunits that are responsible for fast-inactivating A-type K(+) current. MAIN METHODS: Expression pattern of Kv α-subunit, ß-subunit and auxiliary subunit was investigated using immunohistochemistry, in situ hybridization and RT-PCR technique. KEY FINDINGS: The two pore-forming subunits Kv4.1 and Kv4.3 show distinct cellular distributions: Kv4.3 is predominantly in small-sized C-fiber neurons, whereas Kv4.1 is seen in DRG neurons in various sizes. Furthermore, the two classes of Kv4 channel auxiliary subunits are also distributed in different-sized cells. KChIP3 is the only significantly expressed Ca(2+)-binding cytosolic ancillary subunit in DRGs and present in medium to large-sized neurons. The membrane-spanning auxiliary subunit DPP6 is seen in a large number of DRG neurons in various sizes, whereas DPP10 is restricted in small-sized neurons. SIGNIFICANCE: Distinct combinations of Kv4 pore-forming and auxiliary subunits may constitute A-type channels in DRG neurons with different physiological roles. Kv4.1 subunit, in combination with KChIP3 and/or DPP6, form A-type K(+) channels in medium to large-sized A-fiber DRG neurons. In contrast, Kv4.3 and DPP10 may contribute to A-type K(+) current in non-peptidergic, C-fiber somatic afferent neurons.

Effects of bladder outlet obstruction on properties of Ca2+-activated K+ channels in rat bladder.

In this study, we investigated the effects of bladder outlet obstruction (BOO) on the expression and function of large conductance (BK) and small conductance (SK) Ca(2+)-activated K(+) channels in detrusor smooth muscle. The bladder from adult female Sprague-Dawley rats with 6-wk BOO were used. The mRNA expression of the BK channel alpha-subunit, beta1-, beta2-, and beta4-subunits and SK1, SK2, and SK3 channels were investigated using real-time RT-PCR. All subunits except for the BK-beta2, SK2, and SK3 channels were predominantly expressed in the detrusor smooth muscle rather than in the mucosa. The mRNA expression of the BK channel alpha-subunit was not significantly changed in obstructed bladders. However, the expression of the BK channel beta1-subunit and the SK3 channel was remarkably increased in obstructed bladders. On the other hand, the expression of the BK channel beta4-subunit was decreased as the severity of BOO-induced bladder overactivity progressed. In detrusor smooth muscle strips from obstructed bladders, blockade of BK channels by iberiotoxin (IbTx) or charybdotoxin (CTx) and blockade of SK channels by apamin increased the amplitude of spontaneous contractions. These blockers also increased the contractility and affinity of these strips for carbachol during cumulative applications. The facilitatory effects elicited by these K(+) channel blockers were larger in the strips from obstructed bladders compared with control bladders. These results suggest that long-term exposure to BOO for 6 wk enhances the function of both BK and SK types of Ca(2+)-activated K(+) channels in the detrusor smooth muscle to induce an inhibition of bladder contractility, which might be a compensatory mechanism to reduce BOO-induced bladder overactivity.

Comparative studies of ZD0947, a novel ATP-sensitive K(+) channel opener, on guinea pig detrusor and aortic smooth muscles.

The effects of ZD0947, a novel urinary bladder selective ATP-sensitive potassium channel (K(ATP) channel) opener, on carbachol-induced contractions of isolated guinea pig urinary bladder strips were investigated to compare its ability to relax norepinephrine-induced contraction of the aorta. Electrophysiological techniques were also utilized to compare the effects of ZD0947 on membrane currents between guinea pig detrusor and aortic myocytes. ZD0947 caused a significant reduction of the carbachol-induced contractile activity, demonstrating a biphasic relaxation (the first and second components). Although glibenclamide antagonized the effects of two components for the ZD0947-induced relaxation, gliclazide, a selective sulphonylurea receptor 1 (SUR1) antagonist, reduced the effects of the first component but not the second component of the ZD0947-induced relaxation. ZD0947 also reduced the norepinephrine-induced contraction of the aorta. ZD0947 reduced electrical excitability of detrusor smooth muscles, inhibiting spike discharges and also hyperpolarizing the membrane as measured with microelectrodes. In conventional whole-cell configuration, ZD0947 caused a glibenclamide-sensitive K(+) current (i.e., K(ATP) current) at a holding potential of -60 mV in guinea pig detrusor and aortic myocytes. The current density of ZD0947-induced K(ATP) currents in guinea pig detrusor myocytes was significantly larger than that in aortic smooth muscle cells. These results show that ZD0947 caused a significant relaxation through the activation of K(ATP) channels in detrusor muscle.

Association among the symptoms, quality of life and urodynamic parameters in patients with improved lower urinary tract symptoms following a transurethral resection of the prostate.

AIMS: To investigate the associations of symptoms and the quality of life (QOL) with objective variables in a strictly selected large cohort of subjects with symptomatic benign prostatic obstruction (BPO). METHODS: A retrospective study was conducted in 557 males with BPO in whom a symptomatic improvement had been achieved by transurethral resection of the prostate (TURP), thus suggesting that their lower urinary tract symptoms were primarily due to BPO. The association between the preoperative International Prostate Symptom Score (IPSS) and QOL score with objective variables including the residual volumes, prostate size and urodynamic parameters was statistically analyzed. RESULTS: Maximum flow rate (Q(max)) positively and a residual urine volume (PVR) negatively correlated with symptoms and QOL score. Detrusor overactivity (DO) also was weakly, but broadly associated with the symptoms. Degree of detrusor contractility and bladder capacity had a weak association with only some storage symptoms. The degree of bladder outlet obstruction (BOO) positively related to the scores on urgency, straining and total IPSS. Patients' age had positive correlation with the score on nocturia. The prostate volume was only negligibly correlated with either any symptoms or the QOL score. CONCLUSIONS: Parameters, such as Q(max) or PVR, obtained from the noninvasive urodynamics were most widely correlated with symptoms and QOL. Despite a large group with strict selection of men with LUTS possibly relating to BPO being studied, only weak association between the symptoms or QOL and objective parameters including urodynamics was confirmed.

Therapeutic receptor targets for lower urinary tract dysfunction.

The functions of the lower urinary tract, to store and periodically release urine, are dependent on the activity of smooth and striated muscles in the bladder, urethra, and external urethral sphincter. During urine storage, the outlet is closed, and the bladder smooth muscle is quiescent. When bladder volume reaches the micturition threshold, activation of a micturition center in the dorsolateral pons (the pontine micturition center) induces a bladder contraction and a reciprocal relaxation of the urethra, leading to bladder emptying. During voiding, sacral parasympathetic (pelvic) nerves provide an excitatory input (cholinergic and purinergic) to the bladder and inhibitory input (nitrergic) to the urethra. These peripheral systems are integrated by excitatory and inhibitory regulation at the levels of the spinal cord and the brain. Injury or diseases of the nervous system, as well as drugs and disorders of the peripheral organs, can produce lower urinary tract dysfunction. In the overactive bladder (OAB) condition, therapeutic targets for facilitation of urine storage can be found at the levels of the urothelium, detrusor muscles, autonomic and afferent pathways, spinal cord, and brain. There is increasing evidence showing that the urothelium has specialized sensory and signaling properties including: (1) expression of nicotinic, muscarinic, tachykinin, adrenergic, bradykinin, and transient receptor potential (TRP) receptors, (2) close physical association with afferent nerves, and (3) ability to release chemical molecules such as adenosine triphosphate (ATP), acetylcholine, and nitric oxide. Increased expression and/or sensitivity of these urothelial-sensory molecules that lead to afferent sensitization have been documented as possible pathogenesis of OAB. Targeting afferent pathways and/or bladder smooth muscles by modulating activity of ligand receptors (e.g., neurokinin, ATP, or beta3-adrenergic receptors) and ion channels (e.g., TRPV1 or K) could be effective to suppress OAB. In the stress urinary incontinence condition, pharmacotherapies targeting the neurally mediated urethral continence reflex during stress conditions such as sneezing or coughing could be effective for increasing the outlet resistance. Therapeutic targets include adrenergic and serotonergic receptors in the spinal cord as well as adrenergic receptors at the urethral sphincter, which can enhance urethral reflex activity during stress conditions and increase baseline urethral pressure, respectively.

RhoA/Rho kinase-mediated Ca2+ sensitization in the contraction of human prostate.

AIMS: The contractile mechanisms of prostatic smooth muscle have been extensively investigated at the receptor level. However, the intracellular mechanisms have not yet been fully elucidated, especially in human tissue. In the present study, we examined the functional role of RhoA/Rho kinase (ROCK), one of the major intracellular molecules involved in smooth muscle contraction, in the contraction of the human prostate. METHODS: Ring preparations made of cultured human prostatic stromal cells (CHPSCs) or fresh human prostatic tissue was used for an isometric tension study. Gene transfer using baculovirus vector and alpha-toxin permeabilized preparations were also used. RESULTS: RhoA, ROCK I and ROCK II proteins were all expressed in CHPSCs and fresh human prostatic tissue. In CHPSCs ring preparations, the contraction induced by endothelin (ET)-1 was enhanced by over-expression of RhoA and inhibited by ROCK inhibitor. In alpha-toxin permeabilized preparations, ET-1 or GTP-gammaS induced an additional contraction at a constant [Ca2+]i, that was inhibited by ROCK inhibitor. In fresh human prostatic tissue, norepinephrine (NE)-induced contraction was inhibited by ROCK inhibitor at a constant [Ca2+]i in alpha-toxin permeabilized preparations. CONCLUSIONS: These results suggested that RhoA/ROCK-mediated Ca2+ sensitization is likely involved in the contraction of the human prostate. The antagonisms of this pathway may thus be useful as an alternative target in the treatment of benign prostatic hyperplasia (BPH).

Different glibenclamide-sensitivity of ATP-sensitive K+ currents using different patch-clamp recording methods.

Electorophysiological and pharmacological properties of the levcromakalim-induced inward ATP-sensitive K+ currents (K(ATP) currents) in pig proximal urethra were investigated by use of two different whole-cell patch-clamp techniques, namely conventional whole-cell and nystatin-perforated patch recordings. In conventional whole-cell configuration, the levcromakalim (100 microM)-induced K(ATP) current decayed by about 30% in 8 min at a holding potential of -50 mV. In contrast, with the nystatin-perforated patch, 96% of the levcromakalim-induced K(ATP) current still remained even after 8 min application of levcromakalim. The peak amplitude of the levcromakalim-induced inward K(ATP) currents in nystatin-perforated patch was approximately half of those observed in conventional whole-cell configuration. When cytosolic extract of pig urethra was included in the pipette solution, approximately 90% of the levcromakalim (100 microM)-induced K(ATP) current remained at 8 min, even after the establishment of conventional whole-cell configuration. In conventional whole-cell configuration, glibenclamide suppressed the levcromakalim-induced K(ATP) currents in a concentration-dependent manner (Ki=175 nM). Inclusion of 1 mM uridine 5'-diphosphate (UDP) in the pipette solution shifted the glibenclamide-sensitivity (Ki=640 nM) to the right in comparison with that in the absence of UDP (i.e., control). In contrast, using nystatin-perforated patch, glibenclamide inhibited the levcromakalim-induced K(ATP) currents with two affinity sites (high-affinity site, Ki1=10 nM; low-affinity site, Ki2=9 microM). The concentration response curves regarding the inhibitory effects of K(ATP) channel pore blockers (Ba2+ and flecainide) on the levcromakalim-induced K(ATP) currents in conventional whole-cell recording nearly overlapped with those in nystatin-perforated patch recording. These results indicate that the glibenclamide-sensitivity of pig urethral K(ATP) channels in nystatin-perforated patch recording was significantly different from that in a conventional whole-cell configuration, and that the glibenclamide-sensitivity may be modified by some cytosolic factor(s).

Multiple actions of U-37883A, an ATP-sensitive K+ channel blocker, on membrane currents in pig urethra.

The effects of U-37883A, a vascular ATP-sensitive K(+) channel (K(ATP) channel) blocker, on membrane currents were investigated in pig urethral myocytes by use of patch-clamp techniques (conventional whole-cell recordings, nystatin-perforated patches and cell-attached configuration). Tension measurement was also performed to study the effects of U-37883A on the levcromakalim-induced urethral relaxation and the urethral resting tone in the absence and presence of Bay K 8644. Although cumulative application of U-37883A produced a concentration-dependent inhibitory effect on the levcromakalim-induced urethral relaxation, U-37883A did not abolish the relaxation. In nystatin-perforated patch recording, K(ATP) currents activated by levcromakalim were inhibited by U-37883A in a concentration-dependent manner (K(i), 4.7 microM). Approximately 10% of the K(ATP) currents still remained even in the presence of 300 microM U-37883A. In cell-attached mode, extracellular application of U-37883A (100 microM) irreversibly inhibited the activity of the levcromakalim-induced K(ATP) channels. In whole-cell configuration, U-37883A suppressed the peak amplitude of voltage-dependent Ba(2+) currents in a concentration- and voltage-dependent manner, and at 30 microM, shifted the steady-state inactivation curve of the Ba(2+) currents to the left at -90 mV. These results demonstrate that U-37883A reduces not only the activities of K(ATP) channels but also voltage-dependent Ca(2+) channels. Therefore, it is not appropriate to define U-37883A as solely a vascular K(ATP) channel blocker.

Effects of U-37883A on intracellular Ca2+ -activated large-conductance K+ channels in pig proximal urethral myocytes.

Kinetic studies of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydrochloride), a vascular ATP-sensitive K+ channel (KATP channel) blocker, were performed on pig urethral myocytes to investigate inhibitory effects on large-conductance intracellular Ca2+ -sensitive K+ channels (i.e., BKCa channels; 225 pS K+ channels) by use of single-channel recordings (outside-out and inside-out configuration). BKCa channels in pig urethral smooth muscles showed extracellular iberiotoxin (300 nM) sensitivity and voltage dependency. The alpha subunit of BKCa channel proteins was detected in the membrane fraction by use of Western blot technique. Application of U-37883A (> or =10 microM) reduced the activity of BKCa channels in a concentration-dependent manner, not only by decreasing mean openlife time but also by prolonging the mean closed time. These results shows that U-37883A affects channels other than the vascular KATP channel, and demonstrates how it inhibits the activities of BKCa channels in urethral smooth muscles.