Regulation of inwardly rectifying K(+) channel in cultured opossum proximal tubule cells by protein phosphatases 1 and 2A

The inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cells is activated at least in part by protein kinase A (PKA). In this study, we examined the effects of protein serine/threonine phosphatase types 1 (PP-1) and 2A (PP-2A) on activity of the K(+) channel using the patch-clamp technique. In cell-attached patches, channel activity was enhanced by the application of okadaic acid (OA, 1 microM), a membrane-permeable inhibitor of PP-1 and PP-2A, to the bath solution. This enhancement was abolished by the pretreatment of cells with KT5720 (200 nM), a specific inhibitor of PKA. In inside-out patches, channel activity which could be maintained in the presence of ATP (3 mM) in the bath solution was also increased by the addition of OA (1 microM), and the OA-induced increase in channel activity was partially prevented in the presence of KT5720 (200 nM). Direct application of either PP-1 (1 U/ml) or PP-2A (1 U/ml) to the cytoplasmic surface of the patch membrane inhibited channel activity maintained by ATP (3 mM) in inside-out patches. Moreover, channel activity stimulated by PKA (20 nM) in the presence of ATP (3 mM) was also inhibited by the application of either PP-1 (1 U/ml) or PP-2A (1 U/ml). These results indicate that the OA-sensitive protein phosphatase is involved in the regulation of channel activity, and suggest that both PP-1 and PP-2A are candidates responsible for the inhibition of channel activity through dephosphorylation of the PKA-mediated protein phosphorylation.     

Evidence for the presence of a histaminergic neuron system in the rat brain: an immunohistochemical analysis

The binding of calcium antagonists in the rat hippocampal formation was studied using autoradiography. Hippocampal slices were labeled in vitro with [3H]PN 200-110. High densities of binding sites for calcium antagonists were found in the molecular layer of the dentate gyrus and in the CA3 subfield of the hippocampus. After ablation of the granule cells by local injection of colchicine a marked decrease in the number of [3H]PN 200-110 binding sites density was observed on these areas, while binding to other parts of the hippocampal formation and brain was spared. These results strongly suggest the localization of high densities of calcium channels to the granule cells of the dentate gyrus.     

Development of analysis of drugs and toxic substances that can play an immediate role in emergency medical service: what is to be analyzed?

Our laboratory was capable of analyzing less than 20 drugs and toxic substances at the time of the establishment of the Center in 1994. Since the poisoning crimes in 1998, such as the curry poisoning with arsenic in Wakayama, the sodium azide poisoning in Niigata, and the potassium cyanide poisoning in Nagano, we have introduced methods for rapid qualitative analysis of arsenic compounds, cyanides and azides, and developed methods for qualitative analysis of three types of surfactants (cationic, anionic, and nonionic) on the basis of the statistics for intoxication patients transferred to the Center. In 1999, the Analysis Method Investigation Committee of the Japanese Society for Clinical Toxicology requested individual medical institutions to analyze 15 selected intoxicating substances, focusing on the following three aspects. 1. Intoxication with a high degree of fatality. 2. Intoxication where analysis plays an immediate role in treatment. 3. Intoxication with a high frequency of requests by clinical physicians for analysis. The selected substances included methanol, barbital drugs, benzodiazepines, tricyclic or tetracyclic antidepressants, methamphetamine, acetaminophen, salicylic acid, bromovalerylurea, organophosphorus pesticides, carbamate pesticides, paraquat, glufosinate, cyanides, arsenic, and theophylline. Responding to the Committee's request, out laboratory has been making efforts so that analysis of drugs and intoxicating substances can play an immediate role in emergency medical service, giving the highest priority to the aforementioned 15 substances. As a result, anyone of us can now rapidly analyze about 35 substances, including those listed by the Society, day and night.     

Disinhibition in the monkey prefrontal cortex, by injecting bicuculline, induces forelimb movements learned in a GO/NO-GO task

Local injection of a GABA (gamma-aminobutyric acid) antagonist, bicuculline, into Brodmann's area 8 (Walker's areas 8A and 45) of the monkey prefrontal cortex induced movements of the contralateral forelimb in 4 monkeys that were well-trained to perform a two-choice visual discrimination GO/NO-GO task by appropriate release of the hand from a lever. But the same procedures of injection in two naive monkeys did not. These results indicate that, during learning of the task, a new structure responsible for learned movement of the forelimb was made in area 8 and was disinhibited by bicuculline and, furthermore, they suggest that inhibitory GABA neurons suppress tonically efferent neurons associated with the initiation of movement.     

Coexistence of peptides (corticotropin releasing factor/neurotensin and substance P/somatostatin) in the bed nucleus of the stria terminalis and central amygdaloid nucleus of the rat

Coexistence of corticotropin releasing factor and neurotensin and also of substance P and somatostatin was demonstrated in the lateral bed nucleus of the stria terminalis and the central amygdaloid nucleus of the rat, by means of a light microscopic mirror method or immunofluorescent double staining. Using the former technique, a major proportion of corticotropin releasing factor-like immunoreactive cells were found to display neurotensin-like immunoreactivity in the dorsal subdivision of the lateral bed nucleus of the stria terminalis and the lateral subdivision of the central amygdaloid nucleus. On the other hand, the immunofluorescent method showed that a significant number of neurons with both substance P- and somatostatin-like immunoreactivity were located in the ventral subdivision of the lateral bed nucleus of the stria terminalis and the medial subdivision of the central amygdaloid nucleus. Distribution patterns of such co-localized peptides may indicate that there are morphological and biochemical similarities between the dorsal subdivision of the lateral bed nucleus of the stria terminalis and the lateral subdivision of the central amygdaloid nucleus, as well as between the ventral subdivision of the lateral bed nucleus of the stria terminalis and the medial subdivision of the central amygdaloid nucleus. Previous studies have demonstrated that peptide-containing neurons in the lateral bed nucleus of the stria terminalis and central amygdaloid nucleus, such as corticotropin releasing factor-, neurotensin-, substance P- and somatostatin-like immunoreactive cells, project to the lower brainstem. The results of the present study suggest that corticotropin releasing factor/neurotensin and substance P/somatostatin neurons may be part of the lateral bed nucleus of the stria terminalis/central amygdaloid nucleus-lower brainstem pathways.     

cAMP-dependent presynaptic regulation of spontaneous glycinergic IPSCs in mechanically dissociated rat spinal cord neurons

Spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with intact glycinergic presynaptic nerve terminals and evoked IPSCs (eIPSCs) in the slice preparation were investigated using nystatin-perforated patch and conventional whole cell recording modes under the voltage-clamp conditions. Trans-ACPD (tACPD) reversibly reduced the mIPSC frequency without affecting the mean amplitude. The effect was mimicked by a specific metabotropic glutamate receptor (mGluR) II subtype agonist, (2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine (L-CCG-I), and a specific mGluRIII subtype agonist, 2-amino-4-phosphonobutyrate (L-AP4). These inhibitory effects on mIPSC frequency were blocked by the specific antagonists for mGluRII, alpha-methyl-1-(2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine and (RS)-alpha-cyclopropyl-4-phosphonophenylglycine. In the slice preparation, eIPSC amplitude and mIPSC frequency were decreased reversibly by L-CCG-I (10(-6) M) and L-AP4 (10(-6) M). In K(+)-free or K(+)-free external solution with Ba(2+) and Cs(+), Ca(2+)-free or Cd(2+) external solution, the inhibitory effect of tACPD on mIPSC frequency was unaltered. Forskolin and 8-Br-cAMP significantly increased presynaptic glycine release, and prevented the inhibitory action of tACPD on mIPSC frequency. Sp-cAMP, however, did not prevent the inhibitory action of tACPD on mIPSC frequency. It was concluded that the activation of mGluRs inhibits glycine release by reducing the action of cAMP/PKA pathway.     

Localization of [F]fluorodeoxyglucose in mouse brain neurons with micro-autoradiography

This is the first study of micro-autoradiography (micro-ARG) for [¹⁸F]2-fluoro-2-deoxy- -glucose ([¹⁸F]FDG). The localization of [¹⁸F]FDG was demonstrated in dendrites of neuron and also in the myelinated axon in mouse normal brain in vivo. The nucleolus was relatively free of label. The counted silver grain numbers in autoradiogram were linearly correlated to the ¹⁸F radioactivities in the specimen. The micro-ARG using positron emitting ¹⁸F is a very time-saving technique with 4 hours exposure compared with the conventional method using ³H- or ¹⁴C-labeled tracers.