Masaru Ishimatsu

Expression of the TRPM8-immunoreactivity in dorsal root ganglion neurons innervating the rat urinary bladder

The neurochemical phenotypes of the transient receptor potential melastatin-8 (TRPM8)-immunoreactive afferent neurons innervating the rat urinary bladder were examined by using a highly sensitive tyramide signal amplification method, combined with wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing. TRPM8-immunoreactivity was detected in a small proportion of the WGA-HRP-labeled bladder afferent neurons in the dorsal root ganglia of the Th13-L1 (1.14%) and the L6-S1 (1.27%), and these neurons were small in size (<600 microm(2)). The 82.6+/-3.8% of the TRPM8-immunoreactive bladder afferent neurons and 80.9+/-1.5% of the total population of the TRPM8-immunoreactive afferent neurons in the observed dorsal root ganglia expressed NF200. On the other hand, the proportions of the co-expression of TRPM8 and nociceptive markers such as calcitonin gene-related peptide (CGRP), transient receptor potential vanilloid-1 (TRPV1), and isolectin B4 (IB4) in the bladder afferent neurons (81.5+/-5.2% for CGRP, 36.1+/-4.0% for TRPV1, and 15.8+/-5.5% for IB4) were higher in comparison to those in the total population of the TRPM8-immunoreactive afferent neurons (21.9+/-2.4% for CGRP, 16.6+/-1.7% for TRPV1, and 5.4+/-0.5% for IB4), although no significant difference existed for IB4. Our results suggest that the TRPM8-expressing bladder afferents should be classified as Adelta-fibers and C-fibers, while some of these afferents may be involved in nociceptive sensations.

Tachykinins cause inward current through NK1 receptors in bullfrog sensory neurons

The effects of tachykinins on primary afferent neurons of bullfrog dorsal root ganglia (DRG) were examined by using whole-cell patch-clamp methods. Neurokinin A (NKA) caused inward current (INKA) in a concentration-dependent manner. Concentration-response curve showed that the EC50 for NKA was 6 nM. The INKA showed strong tachyphylaxis, when NKA was continuously applied for more than 1 min. Substance P (SP) also produced inward current with potency similar to that of NKA. Neurokinin B (NKB) was less effective in producing the inward current. The order of agonist potency was NKA = SP >> NKB. Spantide ([D-Arg1, D-Trp7.9, Leu11]SP), a non-selective peptide antagonist at tachykinin receptors, reduced the tachykinin-induced current. CP-99,994, a selective non-peptide antagonist for neurokinin-1 (NK1) receptor, inhibited the inward currents produced by NKA and SP. The INKA was associated with decrease in K+ conductance. NKA suppressed both a voltage-dependent K+ current, the M-current (IM), and a voltage-independent background K+ current, IK(B). Intracellular dialysis with GTP gamma S (100 nM) or GDP beta S (100 microM) depressed the INKA. Pre-treatment of DRG neurons with pertussis toxin (PTX) did not prevent the INKA. Depletion of intracellular ATP depressed the INKA. These results suggest that the tachykinin-induced inward current is mediated through the NK1 receptor which mainly couples to PTX-insensitive G-protein in bullfrog primary afferent neurons.

Protective role of heparin/heparan sulfate on oxalate-induced changes in cell morphology and intracellular Ca2+

Alterations in intracellular Ca2+ ([Ca2+]i) are generally associated with cellular distress. Oxalate-induced cell injury of the renal epithelium plays an important role in promoting CaOx nephrolithiasis. However, the degree of change in intracellular free calcium ions in renal epithelial cells during oxalate exposure remains unclear. The aim of this study is to determine whether acute short-term exposure to oxalate produces morphological changes in the cells, induces a change in cytosolic Ca2+ levels in renal tubular epithelial cells and whether the application of extracellular glycosaminoglycans (GAGs) prevents these changes. Cultured Mardin-Darby canine kidney cells were exposed to oxalate, and changes in cytosolic Ca2+ were determined under various conditions. The effect of heparin and heparan sulfate (HS) during oxalate exposure was examined. The change in the GAG contents of the culture medium was also determined. Transmission electron microscopy (TEM) was performed for morphological analysis. The degree of change in cytosolic Ca2+ strongly correlated with oxalate concentration. Cytosolic Ca2+ levels decreased in parallel with an increase in the concentration of oxalate. However, this decrease was strongly inhibited by pretreatment with heparin or HS. TEM revealed cytoplasmic vacuolization, the appearance of flocculent material and mitochondrial damage after oxalate exposure. On the other hand, pretreatment with heparin or HS completely blocked these morphological changes. The present data suggest that acute exposure to a high concentration of oxalate challenges the renal cells, diminishes their viability and induces changes in cytosolic Ca2+ levels. Heparin and HS, which are known as potent inhibitors of CaOx crystallization, may also prevent oxalate-induced cell changes by stabilizing the cytosolic Ca2+ level.

Function and expression pattern of TRPM8 in bladder afferent neurons associated with bladder outlet obstruction in rats

We investigated the function and expression pattern of the transient receptor potential melastatin-8 (TRPM8) in urinary bladder afferent neurons from control and bladder outlet obstruction (BOO) rats. BOO was produced and, after six weeks, the effects of intravesical infusion of menthol, the agonist of TRPM8, were investigated using unanesthetized cystometry. The intravesical infusion of menthol produced an increase in the micturition pressure in both sham surgery and BOO rats. In BOO rats, increased basal and threshold pressure and a decreased micturition interval were observed. Next, the population of TRPM8-positive and the co-expression proportion of TRPM8 with neurochemical markers (NF200 or TRPV1) in the bladder afferent neurons were each compared between the control and BOO rats using retrograde tracing and immunohistochemistry. The population of TRPM8-immunoreactive bladder afferent neurons was larger in BOO rats (3.28±0.43%) than in the control rats (1.33±0.18%). However, there were no statistical differences between the control and BOO rats in the co-expression proportion of neither TRPM8-NF200 (84.1±4.3% vs 79.7±2.7%, p=0.41) nor TRPM8-TRPV1 (33.3±3.6% vs 40.8±2.6%, p=0.08) in the bladder afferent neurons. The present results suggest that the neuronal input through TRPM8-positive bladder afferent neurons are augmented after BOO, however, the neurochemical phenotype of the up-regulated TRPM8-positive bladder afferent neurons is not changed after BOO.

Substance P produces an inward current by suppressing voltage-dependent and -independent K+ currents in bullfrog primary afferent neurons

A whole-cell patch-clamp study was carried out to examine the effect of substance P (SP) on the excitability of neurons in bullfrog dorsal root ganglia (DRG). SP (3 nM to 1 microM) produced an inward current associated with decreased membrane conductance at voltage range between -10 and -130 mV. Neurokinin A (NKA) and neurokinin B (NKB) also produced the inward current in DRG cells; the rank order of agonist potency was NKA = SP much greater than NKB. An antagonist for SP receptors, [D-Arg1, D-Trp7,9, Leu11]SP, did not prevent the response to SP. SP (3 nM to 1 microM) suppressed the voltage-dependent non-inactivating K+ current, the M-current (IM) by reducing the maximum M-conductance. A voltage-independent background K+ current, IK(B), could be recorded at a hyperpolarizing voltage (< or = -60 mV) from DRG neurons. SP (3 nM to 1 microM) produced the inward current associated with decreased IK(B) at a holding potential more negative than -60 mV. The SP-induced inward current reversed its polarity at the equilibrium potential for K ions. Intracellular dialysis with Cs+ blocked the SP-induced responses. Depletion of intracellular ATP reduced SP-induced inward current. These results suggest that the SP-induced inward current was due to suppression of both the IM and IK(B) that are regulated by intracellular activity of ATP in bullfrog DRG neurons.

Persistent α1-adrenergic receptor function in the nucleus locus coeruleus causes hyperexcitability in AD/HD model rats.

Spontaneously hypertensive rats (SHR) are widely used as a model of attention deficit hyperactivity disorder (ADHD) as their ADHD-like behaviors are restored by methylphenidate. However, a postnatal neural development in SHR is unknown. We performed whole cell patch clamp recordings from locus coeruleus (LC) neurons in neonatal [postnatal day (P) 3-5], juvenile (P21-28), and adult (P 49-56) SHR and age-matched Wistar rats to evaluate α1- and α2-adrenergic receptor (ARs) activities at each developmental period. LC neurons in neonatal Wistar rats and SHR showed no difference in resting membrane potential and spontaneous firing rate, while juvenile and adult SHR LC neurons showed depolarized resting membrane potential and faster spontaneous firing rate than in Wistar rats. Blockade of α1-AR activity by prazosin hyperpolarized the membrane and abolished spontaneous firings in all developmental periods in SHR LC neurons, but not in juvenile and adult Wistar rats. α1-AR stimulation by phenylephrine evoked an inward current in juvenile LC neurons in SHR, but not in juvenile Wistar rats. This phenylephrine-induced inward current was abolished by nonselective cation channel blockers. By contrast, α2-AR stimulation-induced outward currents in the presence of an α1-AR antagonist were equivalent in SHR and Wistar LC neurons. These data suggest that Wistar LC neurons lose α1-AR function during development, whereas α1-ARs remain functional in SHR LC neurons. Thus persistent intrinsic activity of α1-ARs may be a neural mechanism contributing to developmental disorders in juvenile SHRs.