Yukio Hayashi

Bladder Overactivity and Hyperexcitability of Bladder Afferent Neurons after Intrathecal Delivery of Nerve Growth Factor in Rats

Nerve growth factor (NGF) has been proposed as an important mediator inducing bladder overactivity under pathological conditions such as spinal cord injury, bladder outlet obstruction, or cystitis. We therefore examined the effects of chronic NGF treatment on bladder activity and the properties of bladder afferent neurons. In adult female rats, NGF (2.5 μg/μl) was infused continuously into the intrathecal space at the L6–S1 level of spinal cord for 1 or 2 weeks using osmotic pumps (0.5 μl/h). Bladder afferent neurons were labeled with axonal transport of Fast Blue injected into the bladder wall. After intrathecal injection of NGF, cystometrograms under an awake condition showed bladder overactivity revealed by time-dependent reductions in intercontraction intervals and voided volume. ELISA analyses showed significant increases in NGF levels in L6–S1 dorsal root ganglia of NGF-treated rats. In patch-clamp recordings, dissociated bladder afferent neurons exhibiting tetrodotoxin (TTX)-resistant action potentials from NGF-treated animals were larger in diameter and had significantly lower thresholds for spike activation compared with sham rats. In addition, the number of TTX-resistant action potentials during 600 ms depolarizing pulses was significantly increased time dependently after 1 or 2 weeks of NGF application. The density of slowly inactivating A-type K+ currents was decreased by 52% in bladder afferent neurons with TTX-resistant spikes after 2 week NGF treatment. These results indicate that increased NGF levels in bladder afferent pathways and NGF-induced reduction in A-type K+ current density could contribute to the emergence of bladder overactivity as well as somal hypertrophy and hyperexcitability of bladder afferent neurons.

Altered expression of potassium channel subunit mRNA and α-dendrotoxin sensitivity of potassium currents in rat dorsal root ganglion neurons after axotomy

Previous studies have raised the possibility that a decrease in voltage-gated K+ currents may contribute to hyperexcitability of injured dorsal root ganglion (DRG) neurons and the emergence of neuropathic pain. We examined the effects of axotomy on mRNA levels for various Kv1 family subunits and voltage-gated K+ currents in L4-L5 DRG neurons from sham-operated and sciatic nerve-transected rats. RNase protection assay revealed that Kv1.1 and Kv 1.2 mRNAs are highly abundant while Kv1.3, Kv1.4, Kv1.5 and Kv1.6 mRNAs were detected at lower levels in L4-L5 DRGs from sham and intact rats. Axotomy significantly decreased Kv1.1, Kv1.2, Kv1.3 and Kv1.4 mRNA levels by approximately 35%, approximately 60%, approximately 40% and approximately 80%, respectively, but did not significantly change Kv1.5 or Kv1.6 mRNA levels. Patch clamp recordings revealed two types of K+ currents in small-sized L4-L5 DRG neurons: sustained delayed rectifier currents elicited from a -40 mV holding potential and slowly inactivating A-type currents that was additionally activated from a -120 mV holding potential. Axotomy decreased both types of K+ currents by 50-60% in injured DRG neurons. In addition, axotomy increased the alpha-dendrotoxin sensitivity of the delayed rectifier, but not slow A-type K+ currents in injured DRG neurons. These results suggest that Kv1.1 and Kv1.2 subunits are major components of voltage-gated K+ channels in L4-L5 DRG neurons and that the decreased expression of Kv1-family subunits significantly contributes to the reduction and altered kinetics of Kv current in axotomized neurons.

Transmembrane interaction mediates complex formation between peptidase homologues and Kv4 channels.

An asthma-related peptidase homologue (DPP10) may act as an auxiliary subunit of Kv4 channels, similar to DPPX. Here we show that DPP10 preferentially binds to Kv4 channel proteins to increase current density and alter channel gating. DPP10 also forms complexes by themselves and with DPPX in the absence of Kv4 channels. DPP10 mRNA is abundantly expressed in nodose and dorsal root ganglia, suggesting that DPP10 participates in controlling airway reactivity and mechanosensation. The region from the N-terminus to the end of the transmembrane of DPP10 mediates its association with the channel, whereas the S1-S2 portion of the channel is sufficient for complex formation. This N-terminal portion of DPP10 also confers all the gating effects produced by the peptidase homologue. Thus, interaction between transmembranes of DPP10/DPPX and Kv4 channel mediates functional complex formation. We call this protein DPPY, instead of DPP10, because of its revealed role as a Kv4 channel regulator.

Bladder hyperactivity and increased excitability of bladder afferent neurons associated with reduced expression of Kv1.4 α-subunit in rats with cystitis

Hyperexcitability of C-fiber bladder afferent pathways has been proposed to contribute to urinary frequency and bladder pain in chronic bladder inflammation including interstitial cystitis. However, the detailed mechanisms inducing afferent hyperexcitability after bladder inflammation are not fully understood. Thus, we investigated changes in the properties of bladder afferent neurons in rats with bladder inflammation induced by intravesical application of hydrochloric acid. Eight days after the treatment, bladder function and bladder sensation were analyzed using cystometry and an electrodiagnostic device of sensory function (Neurometer), respectively. Whole cell patch-clamp recordings and immunohistochemical staining were also performed in dissociated bladder afferent neurons identified by a retrograde tracing dye, Fast Blue, injected into the bladder wall. Cystitis rats showed urinary frequency that was inhibited by pretreatment with capsaicin and bladder hyperalgesia mediated by C-fibers. Capsaicin-sensitive bladder afferent neurons from sham rats exhibited high thresholds for spike activation and a phasic firing pattern, whereas those from cystitis rats showed lower thresholds for spike activation and a tonic firing pattern. Transient A-type K(+) current density in capsaicin-sensitive bladder afferent neurons was significantly smaller in cystitis rats than in sham rats, although sustained delayed-rectifier K(+) current density was not altered after cystitis. The expression of voltage-gated K(+) Kv1.4 alpha-subunits, which can form A-type K(+) channels, was reduced in bladder afferent neurons from cystitis rats. These data suggest that bladder inflammation increases bladder afferent neuron excitability by decreasing expression of Kv1.4 alpha-subunits. Similar changes in capsaicin-sensitive C-fiber afferent terminals may contribute to bladder hyperactivity and hyperalgesia due to acid-induced bladder inflammation.

Roles of Peripheral and Central Nicotinic Receptors in the Micturition Reflex in Rats

PURPOSE We investigated the effects of nicotinic acetylcholine receptor activation in the bladder and central nervous system on the micturition reflex in urethane anesthetized rats. MATERIALS AND METHODS The effects of nicotinic acetylcholine receptor activation on bladder activity were examined during continuous infusion cystometrogram. Nicotine with or without the nicotinic acetylcholine receptor antagonist mecamylamine (Sigma Chemical Co., St. Louis, Missouri) was administered intravesically, intrathecally or intracerebroventricularly in normal or capsaicin pretreated rats. We also examined nicotine induced responses in dissociated bladder afferent neurons from L6 to S1 dorsal root ganglia that were sensitive to capsaicin using whole cell patch clamp recordings. RESULTS Intravesical nicotine (1 to 10 mM) significantly decreased intercontraction intervals in dose dependent fashion. This excitatory effect was abolished by co-application of mecamylamine (3 mM) as well as by capsaicin pretreatment. On patch clamp recordings 300 muM nicotine evoked rapid inward currents that were antagonized by mecamylamine in capsaicin sensitive bladder afferent neurons. Intrathecal and intracerebroventricular administration of nicotine (10 mug) decreased and increase intercontraction intervals, respectively. Each effect was antagonized by mecamylamine (50 mug) administered intrathecally and intracerebroventricularly. The spinal excitatory effect was significantly inhibited by the N-methyl-D-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (20 mug) given intrathecally or by capsaicin pretreatment, although the effects of capsaicin pretreatment were significantly smaller than those of (+)-MK-801 hydrogen maleate. CONCLUSIONS These results indicate that nicotinic acetylcholine receptor activation in capsaicin sensitive C-fiber afferents in the bladder can induce detrusor overactivity. In the central nervous system nicotinic acetylcholine receptor activation in the spinal cord and brain has an excitatory and an inhibitory effect on the micturition reflex, respectively. In addition, the nicotine induced spinal excitatory effect may be mediated by the activation of glutamatergic mechanisms.

Characterization of hyperpolarization-activated current (Ih) in dorsal root ganglion neurons innervating rat urinary bladder.

Afferent pathways innervating the urinary bladder consist of myelinated Adelta-fibers and unmyelinated C-fibers. Normal voiding is dependent on mechanoceptive Adelta-fiber bladder afferents that respond to bladder distention. However, the mechanisms for controlling the excitability of Adelta-fiber bladder afferents are not fully understood. We therefore used whole cell patch-clamp techniques to investigate the properties of hyperpolarization-activated, cyclic nucleotide-gated (HCN) currents (I(h)) in dorsal root ganglion (DRG) neurons innervating the urinary bladder of rats. The neurons were identified by axonal tracing with a fluorescent dye, Fast Blue, injected into the bladder wall. Hyperpolarizing voltage step pulses from -40 to -130 mV produced voltage- and time-dependent inward I(h) currents in bladder afferent neurons. The amplitude and current density of I(h) at a holding potential of -130 mV was significantly larger in medium-sized bladder afferent neurons (diameter: 37.8 +/- 0.3 microm), a small portion (19%) of which were sensitive to capsaicin (1 microM), than in uniformly capsaicin-sensitive small-sized (27.6 +/- 0.5 microm) bladder neurons. In medium-sized bladder neurons, a selective HCN channel inhibitor, ZD7288, dose-dependently inhibited I(h) currents. ZD7288 (10 microM) also increased the time constant of the slow depolarization phase of spike after-hyperpolarization from 91.8 to 233.0 ms. These results indicate that I(h) currents are predominantly expressed in medium-sized bladder afferent neurons innervating the bladder and that inhibition of I(h) currents delayed recovery from the spike after-hyperpolarization. Thus, it is assumed that I(h) currents could control excitability of mechanoceptive Adelta-fiber bladder afferent neurons, which are usually capsaicin-insensitive and larger in size than capsaicin-sensitive C-fiber bladder afferent neurons.

Beneficial effects of suplatast tosilate (IPD-1151T) in a rat cystitis model induced by intravesical hydrochloric acid

To examine the effects of suplatast tosilate (IPD‐1151T), a Th2 cytokine inhibitor recently recognized to improve the symptoms in patients with interstitial cystitis (IC), in a rat model of HCl‐induced chronic cystitis, to elucidate the possible mechanisms by which the drug improves the symptoms of IC.

BALB/c- Fcgr2b −/− Pdcd1 −/− mouse expressing anti-urothelial antibody is a novel model of autoimmune cystitis

We report the impact of anti-urothelial autoantibody (AUAb) on urinary bladder phenotype in BALB/c mice deficient of the FcγRIIb and PD-1. AUAb was present in serum samples from approximately half of the double-knockout (DKO) mice, as detected by immunofluorescence and immunoblots for urothelial proteins including uroplakin IIIa. The AUAb-positive DKO mice showed degeneration of urothelial plaque and umbrella cells, along with infiltration of inflammatory cells in the suburothelial layer. TNFα and IL-1β were upregulated in the bladder and the urine of AUAb-positive DKO mice. Voiding behavior of mice was analyzed by the Voided Stain on Paper method. 10-week-old and older AUAb-positive DKO mice voided significantly less urine per void than did wild type (WT) mice. Furthermore, administration of the AUAb-containing serum to WT mice significantly reduced their urine volume per void. In summary, this report presents a novel comprehensive mouse model of autoimmune cystitis.