Toshihisa Tomoda

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).

Actions of ZD0947, a novel ATP‐sensitive K+ channel opener, on membrane currents in human detrusor myocytes

ATP‐sensitive K+ channels (KATP channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)‐induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated.

The effects of flavoxate hydrochloride on voltage-dependent L-type Ca2+ currents in human urinary bladder

The effects of flavoxate hydrochloride (Bladderon®, piperidinoethyl‐3‐methylflavone‐8‐carboxylate; hereafter referred as flavoxate) on voltage‐dependent nifedipine‐sensitive inward Ba2+ currents in human detrusor myocytes were investigated using a conventional whole‐cell patch‐clamp. Tension measurement was also performed to study the effects of flavoxate on K+‐induced contraction in human urinary bladder. Flavoxate caused a concentration‐dependent reduction of the K+‐induced contraction of human urinary bladder. In human detrusor myocytes, flavoxate inhibited the peak amplitude of voltage‐dependent nifedipine‐sensitive inward Ba2+ currents in a voltage‐ and concentration‐dependent manner (Ki=10 μM), and shifted the steady‐state inactivation curve of Ba2+ currents to the left at a holding potential of −90 mV. Immunohistochemical studies indicated the presence of the α1C subunit protein, which is a constituent of human L‐type Ca2+ channels (CaV1.2), in the bundles of human detrusor smooth muscle. These results suggest that flavoxate caused muscle relaxation through the inhibition of L‐type Ca2+ channels in human detrusor.

The actions of azelnidipine, a dihydropyridine-derivative Ca antagonist, on voltage-dependent Ba2+ currents in guinea-pig vascular smooth muscle

Although azelnidipine is used clinically to treat hypertension its effects on its target cells, Ca2+ channels, in smooth muscle have not been elucidated. Therefore, its effects on spontaneous contractions and voltage‐dependent L‐type Ca2+ channels were investigated in guinea‐pig portal vein.

Mefenamic acid as a novel activator of L-type voltage-dependent Ca2+ channels in smooth muscle cells from pig proximal urethra

1 The effects of mefenamic acid and Bay K 8644 on voltage‐dependent nifedipine‐sensitive inward Ba2+ currents in pig urethral myocytes were investigated by use of conventional whole‐cell configuration patch clamp. 2 Mefenamic acid increased the peak amplitude of voltage‐dependent nifedipine‐sensitive inward Ba2+ current without shifting the position of the current–voltage relationship. 3 Mefenamic acid (300 μM) caused little shift in the activation curve although the voltage dependence of the steady‐state inactivation was shifted to more positive potentials by 11 mV in the presence of mefenamic acid. 4 Bay K 8644 (100 nM) enhanced voltage‐dependent nifedipine‐sensitive inward Ba2+ currents in a concentration‐ and voltage‐dependent manner, shifting the maximum of the current–voltage relationship by 10 mV in the hyperpolarizing direction. 5 Bay K 8644 (1 μM) significantly shifted the voltage dependence of the activation curve to more negative potentials by approximately 9 mV although Bay K 8644 caused little shift in the steady‐state inactivation curve. 6 These results indicate that mefenamic acid increased voltage‐dependent nifedipine‐sensitive inward Ba2+ currents through the activation of L‐type Ca2+ channels with different kinetics from those of Bay K 8644 in pig urethral myocytes.

Modification of ATP-sensitive K+ channels by proteolysis in smooth muscle cells from pig urethra

Patch-clamp experiments have been performed to investigate the effects of endoproteases (such as trypsin, carboxypeptidase B) on both membrane currents and unitary currents in isolated smooth muscle cells from pig proximal urethra (conventional whole-cell configuration, cell-attached configuration, and inside-out patches). Application of either trypsin (1 mg/mL) or carboxypeptidase B (0.1 mg/mL) to the intracellular surface of the excised membrane patches stimulated the activity of a 2.1 pA K+ channel (in symmetrical 140 mM K+ conditions) at a holding potential of -50 mV. The trypsin-induced K+ channels in inside-out configuration exhibited the same amplitude and similar channel opening kinetics to the levcromakalim-induced ATP-sensitive K+ channel (i.e. K ATP channel) in cell-attached patches of the same membrane; however, the sensitivity of the channels to glibenclamide was greatly reduced after the trypsin-treatment. The activity of the trypsin-induced K+ channel was reversibly inhibited by cibenzoline in an inside-out configuration (Ki = 5 microM). It is concluded that trypsin and carboxypeptidase B reactivate the channel with an intact pore activity but the different pharmacological properties of the channels may reflect some change in the conformation in channel proteins after proteolysis.

Predictive factors of late biochemical recurrence after radical prostatectomy

Objective To assess the characteristics of biochemical recurrence in the late period (>5 years after radical prostatectomy) and the differences in the predictors of biochemical recurrence in different periods, we conducted a multicenter retrospective study. Methods We reviewed 478 men who underwent radical prostatectomy for clinically localized prostate cancer. All of the patients were followed up for at least 5 years. The cohort was then divided into three groups; no recurrence group, recurrence <5 years after surgery group and recurrence ≥5 years after surgery group. The background characteristics of each group were compared using the χ2 test. A Cox multivariate regression analysis was performed to determine the predictors of biochemical recurrence in each period. Results Biochemical recurrence occurred in 135 men. In 113 (84%) of the patients, biochemical recurrence occurred at <5 years after surgery; in 22 (16%), it occurred at ≥5 years after surgery. The proportion of men with a low preoperative prostate-specific antigen level was significantly larger in the latter group (P = 0.0023). A preoperative prostate-specific antigen level and a positive surgical margin were significant predictors of biochemical recurrence at <5 years after surgery (hazard ratio: 1.03 and 3.20). A positive surgical margin was also a significant predictor of biochemical recurrence at ≥5 years after surgery (hazard ratio: 3.03); however, a high preoperative prostate-specific antigen level was not. Conclusions Biochemical recurrence occurred at ≥5 years after surgery in 16% of the patients. A positive surgical margin predicted biochemical recurrence in both the early and late periods.