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.

Bladder hyperactivity and increased excitability of bladder afferent neurons associated with reduced expression of Kv1.4 alpha-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.

Differential expression of functional cannabinoid receptors in human bladder detrusor and urothelium.

PURPOSE: Although cannabinoid receptor expression has been demonstrated in human brain and other peripheral neuronal tissues, definitive expression of these receptors in the human bladder has not been reported. Consequently we investigated the expression of functional cannabinoid 1 and 2 receptors in human bladder detrusor and urothelium. MATERIALS AND METHODS: Human bladders were micro-dissected for detrusor (6) and urothelium (8), and analyzed for cannabinoid 1 and 2 mRNA expression using real-time quantitative polymerase chain reaction, and for protein expression using immunohistochemistry and Western blot. Functional response of these receptors was tested by studying the effect of selective cannabinoid 1 and 2 agonists on nerve evoked smooth muscle contraction. RESULTS: Quantitative polymerase chain reaction analysis revealed differential expression of cannabinoid 1 and 2 receptors in detrusor and urothelium. The expression of cannabinoid 1 and 2 receptor mRNA in urothelium was approximately 2-fold higher than in detrusor, although this was not significant (p >0.05). Cannabinoid 1 receptor mRNA expression was significantly higher than cannabinoid 2 receptor expression in the 2 tissue subtypes (p

Expression of hyperpolarization-activated cyclic nucleotide-gated cation channels in rat dorsal root ganglion neurons innervating urinary bladder.

Afferent pathways innervating the urinary bladder consist of myelinated Adelta- and unmyelinated C-fibers, the neuronal cell bodies of which correspond to medium and small-sized cell populations of dorsal root ganglion (DRG) neurons, respectively. Since hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel currents have been identified in various peripheral sensory neurons, we examined the expression of isoforms of HCN channels in the L6-S1 spinal cord and bladder afferent neurons from L6-S1 DRG in rats. Among HCN-1, HCN-2 and HCN-4 channel subtypes, positive staining with HCN-2 antibodies was found in the superficial dorsal horn of the spinal cord and small- and medium-sized unidentified DRG neurons. In dye-labeled bladder afferent neurons, HCN-2-positive cells were found in approximately 60% of neurons, and HCN-2 was expressed in both small- and medium-sized neurons with a higher ratio (expression ratio: 61% and 50% of neurons, respectively) compared with unidentified DRG neurons, in which the HCN expression ratio was 47% and 21% of small- and medium-sized cells, respectively. These results suggest that HCN-2 is the predominant subtype of HCN channels, which can control neuronal excitability, in small-sized C-fiber and medium-sized Adelta fiber DRG neurons including bladder afferent neurons, and might modulate activity of bladder afferent pathways controlling the micturition reflex.

Histological and electrical properties of rat dorsal root ganglion neurons innervating the lower urinary tract.

We investigated whether primary afferent neurons innervating different regions of the lower urinary tract have different histochemical and electrophysiological properties. Neurons in rat L6-S1 DRG were identified by axonal transport of a fluorescent dye. Neurofilament-negative C-fiber cells comprise approximately 70% of bladder and proximal urethral afferent neurons that send axons through the pelvic nerves, but comprise a smaller proportion (51%) of distal urethral neurons that send axons through the pudendal nerves. Isolectin-B4 (IB4) binding was detected in a higher percentage (49%) of C-fiber neurons innervating the distal urethra than in those innervating the bladder or proximal urethra (18-22%). Neurofilament-positive A-fiber neurons innervating the distal urethra had a larger average somal size than neurons innervating the bladder or proximal urethra. In patch-clamp recordings, the majority (70%) of bladder and proximal urethral neurons were sensitive to capsaicin and exhibited TTX-resistant, high-threshold action potentials, whereas a smaller proportion (53%) of distal urethral neurons exhibited TTX-resistant spikes. T-type Ca2+ currents were observed in 47% of distal urethral neurons with TTX-sensitive spikes, but not in TTX-sensitive bladder or proximal urethral neurons. In summary, afferent neurons innervating bladder or proximal urethra differ from those innervating distal urethra. The latter, which more closely resemble cutaneous afferent neurons, consist of a smaller number of C-fiber neurons containing a higher percentage of IB4-positive cells and a more diverse population of A-fiber neurons, some of which exhibit T-type Ca2+ channels. These differences may be related to different functions of respective target organs in the lower urinary tract.