Takakazu Yunoki

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 β3-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).

The effects of caffeine on ATP-sensitive K+ channels in smooth muscle cells from pig urethra

The effects of caffeine on both levcromakalim‐induced macroscopic and unitary currents in pig proximal urethra were investigated by the use of patch‐clamp techniques (conventional whole‐cell configuration and cell‐attached configuration). The effects of caffeine were also examined on currents in inside‐out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K+ channel (Kir6.2) subunits (i.e. Kir6.2ΔC36) which form ATP‐sensitive K+ channels (KATP channels). In conventional whole‐cell configuration, the levcromakalim (100 μM)‐induced inward current (symmetrical 140 mM K+ conditions) was inhibited by caffeine (1 mM) at a holding potential of −50 mV. In contrast, ryanodine (10 μM) caused no significant inhibitory effect on the gradual decay of the levcromakalim‐induced current at −50 mV. The amplitude of the 30 μM levcromakalim‐induced current was enhanced by 3‐isobutyl‐1‐methylxanthine (IBMX, 100 μM). In cell‐attached configuration, the levcromakalim‐induced K+ channel openings were inhibited by subsequent application of 10 mM caffeine, decreasing the channel open probability at −50 mV. Reverse transcriptase‐polymerase chain reaction (RT–PCR) analysis revealed the presence of Kir6.2 transcript in pig urethra. Caffeine (3 mM) inhibited the channel activity of Kir6.2ΔC36 expressed in COS7 cells (3 mM caffeine, 65±6%, n=4; 10 mM caffeine, 29±2%, n=4). These results suggest that caffeine can inhibit the activity of KATP channels through a direct blocking effect on the pore‐forming Kir subunit.

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.

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.

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 involvement of L-type Ca2+ channels in the relaxant effects of the ATP-sensitive K+ channel opener ZD6169 on pig urethral smooth muscle

The effects of ZD6169, a novel ATP‐sensitive K+ channel (KATP channel) opener, were investigated on membrane currents in isolated myocytes using patch‐clamp techniques. Tension measurement was also performed to study the effects of ZD6169 on the resting tone of pig urethral smooth muscle. Levcromakalim was more potent than ZD6169 in lowering the resting urethral tone. Relaxation induced by low concentrations of ZD6169 (3 μM) was completely suppressed by additional application of glibenclamide (1 μM). In contrast, glibenclamide (1 – 10 μM) only partially inhibited the relaxation induced by higher concentrations of ZD6169 (10 μM). Bay K8644 (1 μM) reduced the maximum relaxation produced by ZD6169 (10 μM). In whole‐cell configuration, ZD6169 suppressed the peak amplitude of voltage‐dependent Ba2+ currents in a concentration‐ and voltage‐dependent manner, and at 100 μM, shifted the steady‐state inactivation curve of the voltage‐dependent Ba2+ currents to the left at a holding potential of −90 mV. In cell‐attached configuration, open probability of unitary voltage‐dependent Ba2+ channels (27 pS, 90 mM Ba2+) was inhibited by 100 μM ZD6169 and by 10 μM nifedipine. Reverse transcriptase‐polymerase chain reaction (RT – PCR) analysis revealed the presence of the transcript of the α1C subunit of L‐type Ca2+ channels in pig urethra. These results demonstrate that ZD6169 causes urethral relaxation through two distinct mechanisms, activation of KATP channels at lower concentrations and inhibition of voltage‐dependent Ca2+ channels at higher concentrations (about 10 μM).

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, β-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.

Functional involvement of sulphonylurea receptor (SUR) type 1 and 2B in the activity of pig urethral ATP-sensitive K+ channels

We have investigated the possible roles of sulphonylurea receptor (SUR) type 1 and 2B in the activity of pig urethral ATP‐sensitive K+ channels (KATP channels) by use of patch‐clamp and reverse transcriptase–polymerase chain reaction (RT–PCR) techniques. In voltage‐clamp experiments, not only diazoxide, a SUR1 and weak SUR2B activator, but also pinacidil, a selective SUR2 activator, caused an inward current at a holding potential of −50 mV in symmetrical 140 mM K+ conditions. Gliclazide (1 μM), a selective SUR1 blocker, inhibited the 10 μM pinacidil‐induced currents (Ki=177 μM) and the 500 μM diazoxide‐induced currents (high‐affinity site, Ki1=5 nM; low‐affinity site, Ki2=108 μM) at −50 mV. Application of tolbutamide (100 μM) reversibly caused an inhibition of the 500 μM diazoxide‐induced current at –50 mV. MCC‐134, a SUR type‐specific KATP channel regulator (1–100 μM), produced a concentration‐dependent inward K+ current, which was suppressed by the application of glibenclamide at −50 mV. The amplitude of the MCC‐134 (100 μM)‐induced current was approximately 50% of that of the 100 μM pinacidil‐induced currents. Using cell‐attached configuration, MCC‐134 activated a glibenclamide‐sensitive KATP channel which was also activated by pinacidil. RT–PCR analysis demonstrated the presence of SUR1 and SUR2B transcripts in pig urethra. These results indicate that both SUR1 and SUR2B subunits play a functional role in regulating the activity of pig urethral KATP channels and that SUR1 contributes less than 25% to total KATP currents.

Dual action of ZD6169, a novel K+ channel opener, on ATP‐sensitive K+ channels in pig urethral myocytes

The effects of ZD6169, a novel K+ channel opener, on both membrane and unitary currents in pig urethra were investigated using patch‐clamp techniques. Its effect was also examined on currents in inside‐out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K+ channel (Kir6.2) subunits (Kir6.2ΔC36) which form ATP‐sensitive K+ channels (KATP channels). In current‐clamp mode, ZD6169 (10 μM) induced a concentration‐dependent membrane hyperpolarization. Higher concentrations (30 μM) caused a transient membrane hyperpolarization, followed by a gradual membrane depolarization. On removal of ZD6169, an after hyperpolarization was observed. In conventional voltage‐clamp configuration, at −50 mV in symmetrical 140 mM K+ conditions, ZD6169 (100 μM) caused a transient inward current which gradually decayed. Removal of ZD6169 evoked a much larger amplitude K+ current with a similar time course. ZD6169 produced an inward glibenclamide‐sensitive K+ current, demonstrating a bell‐shaped concentration‐response relationship. In cell‐attached configuration in symmetrical 140 mM K+ conditions, ZD6169 (30 μM) activated an KATP channel which was reversibly suppressed by application of glibenclamide. In contrast, ZD6169 (100 μM) inhibited the activity of the levcromakalim‐induced KATP channels. ZD6169 (100 μM) had no significant effect on the channel activity of Kir6.2ΔC36 in inside‐out configuration, although cibenzoline greatly suppressed the channel activity. These results demonstrate that ZD6169 possesses a dual effect on the activity of the KATP channel; activating at low concentration and inhibiting at higher concentration.