Kohtaro Taniyama

GABA, Glutamic Acid Decarboxylase, and GABA Transaminase Levels in the Myenteric Plexus in the Intestine of Humans and Other Mammals

Abstract: Regional distribution of endogenous γ‐ aminobutyric acid (GABA), its synthesizing enzyme, glutamic acid decarboxylase (GAD), and metabolic enzyme, GABA transaminase (GABA‐T), were determined in the intestinal tract of guinea pigs and cats and the findings compared with the number of ganglion cells in Auerbachs plexus. There were positive correlations among the GABA contents and the numbers of neural cells of the plexus. The precise localization of GABA and GAD in individual layers (mucosa, circular and longitudinal muscles, and Auerbachs plexus) in the human and cat colon was also determined. The endogenous GABA contents and GAD activity were the highest in Auerbachs plexus in tissues of both species. These results indicate that GABA is synthesized and localized in Auerbachs plexus and probably plays a significant role in the enteric nervous system.

A phorbol ester and A23187 act synergistically to release acetylcholine from the guinea pig ileum

Electrical stimulation of guinea pig ileum preloaded with [3H]choline provokes the release of [3H]acetylcholine (ACh) in a Ca2+‐dependent manner. This release was markedly increased by the tumor‐promoting phorbol ester, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). The combination of the ionophore A23187 and TPA produced the release of [3H]ACh up to a level equal to or exceeding a maximal response induced by electrical stimulation. A23187 alone gave only a minor response and TPA alone had no apparent effect on the [3H]ACh release. Thus, protein kinase C probably plays a role in cell surface signal transduction related to the release of transmitters from nerve endings.

GABA evoked ACh release from isolated guinea pig ileum

To identify the target cells of GABAergic neurons located in the myenteric plexus, the action of gamma-aminobutyric acid (GABA) on the release of acetylcholine (ACh) and on the contractions was studied using the isolated guinea pig ileum. GABA evoked a release of 3H-ACh from the contracting ileum, under conditions of loading with 3H-choline. As both the GABA-evoked release of 3H-ACh and the contractions were inhibited by bicuculline, tetrodotoxin and furosemide, but not by hexamethonium, this release seems to be evoked through GABA receptors which are bicuculline sensitive and associated with the Cl- ion channel.

Involvement of protein kinase C in the Ca2+-dependent vesicular release of GABA from central and enteric neurons of the guinea pig

The involvement of protein kinase C (PKC) in the release of endogenous gamma-aminobutyric acid (GABA) was studied using slices of deep cerebellar nucleus and strips of small intestine from the guinea pig. 12-O-tetradecanoylphorbol 13-acetate (TPA), but not 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD), potentiated the high K+-evoked release of GABA from both preparations in the presence of tetrodotoxin. Ouabain evoked the release of GABA from both preparations, and this release was not altered by TPA. Therefore, the activation of protein kinase C potentiates the Ca2+-dependent vesicular release of GABA from nerve terminals of the central and enteric GABAergic neurons of the guinea pig.

Inhibition by cyclic AMP of phorbol ester-potentiated norepinephrine release from guinea pig brain cortical synaptosomes.

Abstract: The involvement of Ca2+/pnospholipid‐dependent protein kinase (protein kinase C, PKC) and cyclic AMP‐de‐pendent protein kinase in the K+‐evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12‐O‐Tetradecanoyl‐phorbol‐13‐acetate (TPA), a potent activator of PKC, enhanced the K+‐evoked release of [3H]NE, in a concentration‐dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10−‐7M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA‐induced po‐tentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA‐potentiated NE release. A novel inhibitor of cyclic AMP‐dependent protein kinase, KT5720, blocked both the forskolin‐induced increase in the evoked release and its inhibition of TPA‐induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+‐dependent release of NE by activating cyclic AMP‐dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP‐dependent protein kinase acts negatively on the PKC‐activated exocytotic neu‐rotransmitter release process in brain synaptosomes of the guinea pig.

Presence of γ-aminobutyric acid and glutamic acid decarboxylase in Auerbach's plexus of cat colon

Regional distributions of endogenous gamma-aminobutyric acid (GABA) and ganglion cells in Auerbachs plexus were studied in the gastrointestinal tract of cats. GABA was determined by enzymatic and fluorometric assay. There was a positive correlation between the GABA content and the number of cells in this plexus. We analyzed the precise localization of GABA and glutamic acid-decarboxylase (GAD) in individual layers, mucosa, circular and longitudinal muscles and Auerbachs plexus, from freeze-dried sections of cat colon by the enzymatic cycling method. Both GABA contents and GAD activity in Auerbachs plexus were highest in all 4 layers. Thus, the possibility that GABA is synthesized and localized within Auerbachs plexus has to be considered.

Responses to Adenine Nucleotides and Related Compounds of Isolated Dog Cerebral, Coronary and Mesenteric Arteries

In helically cut strips of dog cerebral, coronary and mesenteric arteries contracted with prostaglandin F2 alpha, adenosine, AMP, ADP, ATP and cyclic AMP produced dose-related, persistent relaxation. On the basis of ED50S, the relaxant effect seen in cerebral arteries was in the order of AMP, ADP, ATP greater than adenosine, cyclic AMP greater than aderine, dibutyryl cyclic AMP greater than inosine. On the basis of maximum relaxations, the effect was in the order of adenine, dibutyryl cyclic AMP greater than or equal to adenosine, AMP, ADP, ATP greater than cyclic AMP, inosine. The relaxant responses to adenosine, AMP, ADP, ATP and cyclic AMP were attenuated by treatment with aminophylline, whereas the relaxations induced by dibutyryl cyclic AMP, adenine and inosine were not altered. It is concluded that adenosine, AMP, ADP and ATP, but not adenine and inosine, appear to share receptors underlying arterial relaxations, and the cyclic AMP-induced relaxation does not derive from increments in cellular cyclic AMP. Treatment with aspirin did not alter the relaxant effect of ADP, adenosine and adenine, suggesting that the release of vasoactive prostaglandins is not involved.

Dual effect of trimebutine on contractility of the guinea pig ileum via the opioid receptors

Preparations of longitudinal muscle attached to myenteric plexus from guinea pig ileum were used to observe the effect of trimebutine on intestinal motility. Electrical stimulation at 0.2 Hz and 5 Hz produced contraction mediated by the release of acetylcholine in the preparations. The response to low-frequency stimulation (0.2 Hz) was inhibited by trimebutine (10(-8)-10(-5) mol/L), and the response to high-frequency stimulation (5 Hz) was enhanced by the drug at low concentrations (10(-8)-10(-7) mol/L) and inhibited by high concentrations (10(-6)-10(-5) mol/L). This enhancement was mimicked by [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, and was antagonized by naloxone but not by MR2266. Enhancement by trimebutine was inhibited by yohimbine. Trimebutine (greater than or equal to 10(-8) mol/L) inhibited stimulation (5 Hz)-evoked release of norepinephrine, and the trimebutine effect was antagonized by naloxone but not by MR2266. Low concentrations of trimebutine inhibit norepinephrine release via the mu-opioid receptor and enhance intestinal motility by preventing the adrenergic inhibition of acetylcholine release. Inhibition by trimebutine was antagonized either by naloxone or MR2266. High concentrations of trimebutine may inhibit acetylcholine release via the mu- and kappa-opioid receptors, after which the intestinal motility is inhibited. Trimebutine at further high concentrations (greater than 10(-5) mol/L) contracted single smooth muscle cells from the circular muscle layers but not from the longitudinal muscle layers. The usual dose of trimebutine may exert dual effect on the intestinal motility indirectly through cholinergic and adrenergic neurons without direct effect on the smooth muscle.

Expression of the GABAB receptor in Xenopus oocytes and inhibition of the response by activation of protein kinase C

The functional GABAB receptor was expressed in Xenopus oocytes by injecting mRNA obtained from the cerebellum of the rat. Application of GABA in the presence of bicuculline induced a hyperpolarization under current‐clamp conditions and an outward current under voltage‐clamp conditions. Baclofen mimicked the effect of GABA in the presence of bicuculline, and the effect of baclofen was antagonized by phaclofen. The GABA‐induced outward current was slightly inhibited by treatment with GDP‐β‐S and was completely inhibited by treatment with GTP‐γ‐S. The activation of protein kinase C by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), but not 4α‐phorbol‐12,13‐didecanoate, suppressed the GABAB receptor‐mediated hyperpolarization, and the effect of TPA was antagonized by sphingosine. Thus, activation of protein kinase C inhibits the expressed GABAB receptor‐mediated response.

Enteric γ-aminobutyric acid-containing neurons and the relevance to motility of the cat colon

Abstract γ-Aminobutyric acid (GABA)-containing neurons were identified and the functional relevance in the motility of the colon was studied. Autoradiography of the cat colon treated with [ 3 H]GABA demonstrated scattered neurons in the myenteric plexus selectively labeled with [ 3 H]GABA. Electrical transmural stimulation of the isolated cat colon led to an increase in the Ca 2+ -dependent, tetrodotoxin-sensitive release of endogenous GABA. γ-Aminobutyric acid increased the amplitude of rhythmic contractions of the circular muscle of the colon and also the release of acetylcholine, which was Ca 2+ -dependent and tetrodotoxin-sensitive. Scopolamine inhibited the GABA-evoked rhythmic contractions, without effect on the evoked release of acetylcholine. Bicuculline and furosemide reduced the amplitude of spontaneous rhythmic contractions and the tone, which was reversed by GABA. These results suggest that GABA-containing neurons are involved in the control of motility of the cat colon, due to the stimulation of cholinergic neurons.