Shingo Tsukada

Expression of a system L neutral amino acid transporter at the blood-brain barrier

Amino acid transport system L has been proposed to be one of the major nutrient transport systems at the blood–brain barrier. Using immunohistochemical analyses, a system L transporter LAT1 was shown to be expressed in the brain capillary endothelial cells in rats. Because LAT1 was co-expressed with 4F2 heavy chain which brings LAT1 to the plasma membrane, LAT1 is proposed to be functional in the plasma membrane of brain capillary endothelial cells. Both LAT1 and 4F2hc immunoreactivities were detected in a double line appearance surrounding endothelial cell nuclei, suggesting both proteins are present in the luminal and abluminal membranes. LAT1 is, thus, a blood–brain barrier system L transporter responsible for the permeation of aromatic or branched-chain amino acids and amino acid-related drugs such as L-DOPA.

High affinity D- and L-serine transporter Asc-1: cloning and dendritic localization in the rat cerebral and cerebellar cortices.

System asc transporter Asc-1, expressed in the brain, transports D- and L-serine with high affinity. To determine the localization of Asc-1 in the rat brain, we isolated a cDNA for the rat orthologue of Asc-1. The encoded protein designated as rAsc-1 (rat Asc-1) exhibited 98% sequence identity to mouse Asc-1 (mAsc-1). Based on amino acid sequences of rAsc-1 and mAsc-1, two polyclonal antibodies against Asc-1 were generated and used for the immunohistochemical analysis on the cerebral and cerebellar cortices of rats and mice. Asc-1 immunoreactivity was detected in neurons, including cerebellar Purkinje neurons and pyramidal neurons in the neocortex and hippocampus. It was clearly localized in dendrites as well as somata. The localization of Asc-1 in brain suggests the significant contribution of Asc-1 to amino acid mobilization in brains including the synaptic clearance of D-serine and the neuronal uptake of L-serine that is essential for survival and dendrite growth of Purkinje neurons in particular.

Activation of protein kinase A by nitric oxide in cultured dorsal root ganglion neurites of the rat, examined by a fluorescence probe, ARII

To study the roles of nitric oxide (NO) in growth of nerve fibers, (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine (NOR3), an NO-donor, was applied to cultured dorsal root ganglion (DRG) neurites from a micropipette. Ejection of a small volume of 1 mM NOR3 solution (not more than 1 pl/s) from a micropipette to terminal branches of neurites caused enlargement of the neurites, and often, elongation of their growth cones. This neurite enlargement was blocked by inhibitors for soluble guanylate cyclase. The neurite enlargement did not occur when protein kinase A (PKA) was inhibited. To prove that NOR3 activated PKA, we introduced a fluorescence peptide probe, ARII that reduces its fluorescence by activated PKA, to monitor PKA activity in DRG neurites. ARII fluorescence was reduced by NOR3, which was not observed when PKA was inhibited by its specific inhibitors. These indicated that PKA was indeed activated by NO. To examine whether the PKA activation is due to inhibition of phosphodiesterase III (PDE III) by cyclic GMP, we applied PDE III-specific inhibitors and found that the inhibitions activated PKA. Since PKA regulates various neuronal functions, our finding that NO activates PKA is important to understand roles of NO in nerve fibers.

Real-time measurement of nitric oxide by luminol-hydrogen peroxide reaction in crystalloid perfused rat heart.

The objective of this study was to develop an assay system that allows continuous monitoring of nitric oxide (NO) released from crystalloid perfused hearts. We utilized chemiluminescence reaction between NO and luminol-H(2)O(2) to quantify the NO level in coronary effluent. Isolated rat hearts were subjected to ordinary Langendorffs perfusion, and the right ventricle was cannulated to sample coronary effluent. After equilibration, the coronary flow rate was set constant and the hearts were paced at 300 bpm. Coronary effluent was continuously sampled and mixed with the chemiluminescent probe containing 0.018 mmol/l luminol plus 10 mmol/l H(2)O(2). Chemiluminescence from the mixture of coronary effluent and the probe was continuously measured. NO concentration was calibrated by various concentrations (0.5-400 pmol/l) of standard NO solution. The lower detection limit of NO was 1 pmol/l. Basal NO release from isolated perfused rat heart was 0.41 +/- 0.17 pmol/min/g of heart weight, and that was significantly suppressed by 0.1 mmol/l of L-NAME to 0.18 +/- 0.10 pmol/min/g of heart weight (n = 7). Application of 0.1 and 0.3 micromol/l acetylcholine increased NO level in the coronary effluent, in a concentration-dependent manner, from 6.6 +/- 1.7 in a baseline condition to 16.3 +/- 7.4 and 30.3 +/- 16.1 pmol/l at each peak, respectively. Thrombin at 1 and 10 U/ml also increased NO level from 17.6 +/- 4.3 in control to 35.5 +/- 10.4 and 48.7 +/- 8.7 pmol/l at each peak, respectively (n = 7). Thus, this assay system is applicable to the continuous real-time measurement of NO released from crystalloid perfused hearts, and it may be useful for the study of physiological or pathophysiological role of NO in coronary circulation.

NT-3, but not NGF or BDNF, inhibited apoptosis of merkel cells in culture

We previously reported that intracerebroventricular administration of ethacrynic acid (EA) to mice induced generalized tonic-clonic convulsions and that expression of c-fos mRNA increased biphasically, with a transient increase at 60min and a prolonged increase on the 7-14th day after EA administration. In the present study, the expression of NGF also showed biphasic increases with close spatiotemporal correlation with c-fos expression. In addition, immunohistochemical analysis showed an apparent decrease in the density of dendrites and the number of cell somata of parvalbumin (PARV)-positive cells, a subpopulation of GABAergic interneurons, in hippocampus on the 7th day after EA injection. Furthermore, intraperitoneal injection with subconvulsive dose of kainic acid evoked severe seizure in 77% of EA-treated mice on the 14th day. These findings suggest that the prolonged increases in c-fos and NGF mRNA expression and the damage of PARV-positive neurons play a crucial role in the development of seizure susceptibility.

Nitric oxide induced neurite extension through cGMP - PKA pathway

Nitric oxide (NO), a diffusible second messenger molecule, has been known to inhibit the growth of dorsal root ganglion (DRG) neurite in culture. We had already reported an evidence that NO promotes growth of neurite if it was applied locally to a growing tip. Here we report that the enhancement of neurite extension induced by NO, might be contributed by cGMP -PKA pathway. Ejection of a NO donor (NOR3) solution from a micropipette to a growth cone induced a rapid enhancement of neurite extension. LY83583 or Methylene blue, inhibitors for guanylate cyclase, suppressed the NO induced neurite extension. Elevation of cGMP at a growth cone by local photo lysis of caged cGMP also induced extension of the neurite. KT5720, an inhibitor of PKA, suppressed both the NO induced and caged cGMP induced neurite extension. We conclude that NO cGMP PKA pathway may contribute to growth of neurite in specific conditions.

1218 Caged cyclic GMP induced neurite elongation of rat DRG neurons

Since the roles of.cyclic GMP on neurite growth have remained unknown, we examined the effects of Photoactivatable cyclic nucleotides on shape and motility of rat Dorsal Root Ganglion (DRG) cells. The cyclic GMP or AMP was loaded into cells and flash photolysised by UV laser scanning microscope (LSM). The responses of the neurites were monitored using time-lapse video microscopy. Both messengers altered growth cone shape and motility. Photolysis of cyclic GMP promoted neurite outgrowth, and filopodial expansion and motility. Moreover, focal cGMP elevation in the branch of neurites promoted the point of neurite extension selectively. Caged CAMP mimicked the effects of cGMP. These findings indicate that cGMP may act as a second messenger in the cytoplasm of the growth cone during neurite elongation.

703 Programmed death of mechano-sensitive merkel cells during monolayer culture in serum-free media

Merkel cells are mechano-receptor cells in the skin that form synaptic contact with primary sensory nerve fibers. Culturing of these cells in vitro has not been successful. We report here that an almost pure culture of Merkel cells in chemically-defined media was achieved, and that these cells underwent apoptosis during culture in these media. In order to obtained Merkel cells, we dissected cores of hair follicles located in the upper-lips of the rat under binocular microscope, and then, they were digested with collagenase in L-15 medium. Merkel cells were isolated from the cores by pippeting, and were platted on poly-L -lysine-coated plastic dishes. Merkel cells were identified by staining with a fluorescence marking dye, quinacrine. During culture in the medium, population of the Merkel cells reduced quickly in time. Addition of serum (5 %) or actinomycin D (2 nM) in the medium prevented the reduction in the cell population. Furthermore, nuclei of many Merkel cells were well stained with TUNEL-method. We thus concluded that the loss of Merkel cells during culture in the serum-free medium was due to the programmed cell death.