Hiromi Hiruma

Low-concentration lidocaine rapidly inhibits axonal transport in cultured mouse dorsal root ganglion neurons.

BackgroundAxonal transport plays a critical role in supplying materials for a variety of neuronal functions such as morphogenetic plasticity, synaptic transmission, and cell survival. In the current study, the authors investigated the effects of the analgesic agent lidocaine on axonal transport in neurites of cultured mouse dorsal root ganglion neurons. In relation to their effects, the effects of lidocaine on the growth rate of the neurite were also examined. MethodsIsolated mouse dorsal root ganglion cells were cultured for 48 h until full growth of neurites. Video-enhanced microscopy was used to observe particles transported within neurites and to measure the neurite growth during control conditions and in the presence of lidocaine. ResultsApplication of 30 &mgr;m lidocaine immediately reduced the number of particles transported in anterograde and retrograde axonal directions. These effects were persistently observed during the application (26 min) and were reversed by lidocaine washout. The inhibitory effect was dose-dependent at concentrations from 0.1 to 1,000 &mgr;m (IC50= 10 &mgr;m). In Ca2+-free extracellular medium, lidocaine failed to inhibit axonal transport. Calcium ionophore A23187 (0.1 &mgr;m) reduced axonal transport in both directions. The inhibitory effects of lidocaine and A23187 were abrogated by 10 &mgr;m KN-62, a Ca2+–calmodulin-dependent protein kinase II inhibitor. Application of such low-concentration lidocaine (30 &mgr;m) for 30 min reduced the growth rate of neurites, and this effect was also blocked by KN-62. ConclusionsLow-concentration lidocaine rapidly inhibits axonal transport and neurite growth via activation of calmodulin-dependent protein kinase II.

IMMUNOHISTOCHEMICAL LOCALISATION OF REGULATORY NEUROPEPTIDES IN HUMAN CIRCUMVALLATE PAPILLAE

The occurrence and distribution of neuropeptide‐containing nerve fibres in the human circumvallate papillae were examined by the peroxidase–antiperoxidase immunolocalisation method using surgical specimens that had not been subjected to radiotherapy, and the abundance of neuropeptide‐containing fibres was expressed as the percentage of total nerve fibres demonstrated by protein gene product (PGP) 9.5 immunoreactivity for a quantitative representation of these peptidergic fibres. Substance P (SP) and calcitonin gene‐related peptide (CGRP) immunoreactive (IR) nerve fibres were densely distributed in the connective tissue core of the circumvallate papillae, and some SP and CGRP‐IR fibres were associated with the taste buds. A moderate number of vasoactive intestinal polypeptide (VIP)‐IR fibres and a few galanin (GAL)‐IR fibres were also seen in the connective tissue core and subepithelial layer. There were, however, no VIP‐IR or GAL‐IR fibres associated with the taste buds. Neuropeptide Y (NPY)‐IR fibres were few and were associated with the blood vessels. Within the epithelium of the circumvallate papillae, no peptidergic fibres were found, although a number of PGP 9.5‐IR fibres were detected. The abundance of SP, CGRP, VIP, and GAL‐IR fibres expressed as the percentage of total PGP 9.5 IR fibres was 25.35±3.45%, 22.18±3.26%, 10.23±1.18%, and 4.12±1.05%, respectively. The percentage of NPY‐IR fibres was below 3%. In a deeper layer of the papillae, a few VIP, GAL, and NPY‐IR ganglion cells were found, and VIP immunoreactivity was detected in a few cells of the taste buds. There was no somatostatin, leucine enkephalin, or methionine enkephalin immunoreactivity in the circumvallate papillae. These results suggest that the dense SP and CGRP‐IR fibres within the connective tissue core of the human circumvallate papillae may be involved in the deep sensation of the tongue.

Effects of substance P and calcitonin gene-related peptide on axonal transport in isolated and cultured adult mouse dorsal root ganglion neurons

Substance P and calcitonin gene-related peptide (CGRP) released from primary sensory neurons are known to play important roles in nociception and nociceptive transmission. In the present study, we attempted to clarify the roles of these neuropeptides in the regulation of axonal transport in sensory neurons. Cells were isolated from adult mouse dorsal root ganglia and cultured in F-12 medium containing fetal bovine serum for 48 h until their neurites were grown. These isolated and cultured DRG cells were mostly (>98%) small (diameter <25 microm) and medium (diameter, 25-40 microm) in size, and were immunoreactive for substance P and CGRP (85.9 and 66. 0% of total cells, respectively). Video-enhanced microscopy was applied to observe particles transported within neurites. Application of substance P (100 nM) decreased the number of particles transported in both anterograde and retrograde directions in each of DRG neurons tested (n=5). The instantaneous velocities of individual particles transported in anterograde and retrograde directions were also reduced by substance P. In contrast, alpha-CGRP (100 nM) increased the number of particles transported in both directions in each of DRG neurons tested (n=5), and also increased the instantaneous velocities of particles transported bidirectionally. Application of beta-CGRP (100-1000 nM) did not elicit any effect on axonal transport. Therefore, axonal transport in sensory neurons seems to be modulated by substance P and alpha-CGRP, both of which can be derived from its own and adjacent sensory neurons.

Inhibitory effect of histamine on axonal transport in cultured mouse dorsal root ganglion neurons

Histamine is important in mediating peripheral sensory information such as inflammation, allergic hypersensitivity, and itch. In the present study, using video-enhanced microscopy, we investigated the effect of histamine on axonal transport in cultured dorsal root ganglion (DRG) neurons of the mouse. Application of histamine (100 microM) reversibly reduced the number of particles transported within neurites in both anterograde and retrograde directions. The histamine H(1)-receptor agonist 2-thiazolylethylamine (100 microM) and the H(3)-receptor agonist R-alpha-methylhistamine (100 microM) also reduced anterograde and retrograde axonal transport, whereas the histamine H(2)-receptor agonist dimaprit (100-1000 microM) had no effect. The effect of histamine was partially blocked by pretreatment with H(1)-receptor antagonist pyrilamine (1 microM) or the H(3)-receptor antagonist thioperamide (1 microM). Pretreatment with a combination of pyrilamine (1 microM) and thioperamide (1 microM) completely blocked the response to histamine. The H(2)-receptor antagonist cimetidine (1 microM) was ineffective. These results suggest that histamine inhibits axonal transport of cultured mouse DRG neurons via the activation of H(1)- and H(3)-receptors.

NEURONAL SYNCHRONIZATION AND IONIC MECHANISMS FOR PROPAGATION OF EXCITATION IN THE FUNCTIONAL NETWORK OF IMMORTALIZED GT1-7 NEURONS : OPTICAL IMAGING WITH A VOLTAGE-SENSITIVE DYE

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage‐sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1–7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1–7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1–7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus‐evoked excitation was prevented by the voltage‐dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage‐dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L‐type) and flunarizine (T‐type>L‐type), but not apparently affected by &ohgr;‐agatoxin IVA (P‐ and Q‐type) and &ohgr;‐conotoxin MVIIA (N‐type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4‐aminopyridine (4‐AP) and prevented by apamin. These results suggest that voltage‐dependent Na+ channels and L‐ and T‐type Ca2+ channels are involved in the propagation of electrical signals in the GT1–7 neuronal network. Ionic mechanisms, through 4‐AP‐ or apamin‐sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.

Effects of Ecabet Sodium, a Novel Gastroprotective Agent, on Mucin Metabolism in Rat Gastric Mucosa

The effects of ecabet sodium (ecabet), 12-sulfodehydroabietic acid monosodium salt, on gastric mucin biosynthesis in rat antrum were compared with those in the corpus. Intragastric administration of ecabet significantly increased [3H]glucosamine incorporation into antral mucin as well as into corpus mucin during five successive hours of organ culture. In contrast, mucin biosynthesis in either antrum or corpus was not susceptible to the addition of ecabet to the culture medium. Ecabet-induced stimulation of prostaglandin E2 production in the antrum was essentially the same as that seen in the corpus. In antrum treated with 100 mg/kg ecabet, immunoreactivity with three distinct anti-mucin monoclonal antibodies was found not only in the specific mucus-producing cells, but also in the secreted mucus present at the surface gel layer. These results suggest that ecabet enhances the mucin metabolism, and this stimulation occurs in both the corpus and antrum, suggesting that ecabet might be a useful tool for the further clarification of the regulatory mechanism of antral mucin synthesis.

Axonal transport is inhibited by a protein kinase C inhibitor in cultured isolated mouse dorsal root ganglion cells

We investigated roles of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein II (CAM II) kinase activities in the maintenance of axonal transport in cultured isolated mouse dorsal root ganglion (DRG) cells. Video-enhanced microscopic recordings revealed that the PKC inhibitor chelerythrine (1 microM) reduced anterograde and retrograde axonal transport, while the CAM II kinase inhibitor KN-62 (10 microM) had no effect. Morphological observation showed that neurite growth was prevented by the presence of chelerythrine (1 microM). From these results, we conclude that PKC activity is required to maintain axonal transport and thereby neurite growth.

EXTRACELLULAR POTASSIUM RAPIDLY INHIBITS AXONAL TRANSPORT OF PARTICLES IN CULTURED MOUSE DORSAL ROOT GANGLION NEURITES

Changes in extracellular potassium concentration ([K+]o) modulate a variety of neuronal functions. However, whether axonal transport, which conveys materials to the appropriate destination for morphogenesis and other neuronal functions, depends on the extracellular K+ environment remains unclear. We therefore examined the effects of changes in [K+]o on axonal transport of particles visualized by video-enhanced microscopy in cultured mouse dorsal root gan-glion neurites. Increases in [K+]o (delta[K+]o > or = 2.5 mM) from control concentration (5 mM) inhibited both anterograde and retrograde axonal transport within a few minutes in a concentration-dependent manner. Conversely, removal of extracellular K+ induced the rapid facilitation of transport in both directions. These inhibitory and facilitatory responses were completely blocked by the K+ channel blocker tetraethylammonium (TEA), suggesting that the effect of changes in [K+]o involves the TEA-sensitive K+ channels. Increases in [K+]o provoked membrane depolarization in the absence and presence of TEA. Another depolarizing agent, veratridine, did not produce an effect on axonal transport. These results suggest that the extracellular K+-mediated inhibition of axonal transport does not depend on membrane depolarization. The inhibitory effect of increasing [K+]o on axonal transport was retained in calcium (Ca2+)-free extracellular medium, indicating that the inhibitory effect of extracellular K+ does not result from Ca2+ influx through voltage-dependent Ca2+ channels. In chloride (CI-)-free medium, increasing [K+]o failed to inhibit axonal transport, implying that the extracellular K+-mediated inhibition of axonal transport may be due to an increase in intracellular Cl- concentration associated with increases in the net inward movement of K+ and CI- across the membrane. Our results suggest that the extracellular K+ environment is involved in the rapid modulation of axonal transport of particles in dorsal root ganglion neurites.