Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfidebonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys 149 , we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in ␤-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys 149 . Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.In both prokaryotic and eukaryotic cells, glyceraldehyde-3-phosphate dehydrogenase (GAPDH 2 ; EC 1.2.1.12) plays a central role in glycolysis, catalyzing the reversible conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate in a reaction that is accompanied by the reduction of NAD ϩ to NADH. Mammalian GAPDH is a homotetramer composed of four identical subunits. Recent studies show that mammalian GAPDH has diverse activities unrelated to its glycolytic function (1, 2), including roles in membrane fusion, microtubule bundling, nuclear RNA transport (2), regulation of Ca 2ϩ homeostasis (3), and transcription (4). In addition to the various functions of GAPDH described above, particular attention is paid to its role in apoptosis (5-7). Although the proapoptotic role(s) of GAPDH seems to depend upon its accumulation in the particulate fractions, including the nucleus (5, 7), the detailed mechanism is still unclear.Recently, it has been suggested that a wide variety of neurodegenerative diseases are characterized by the accumulation of intracellular and extracellular protein aggregates (8,9). An initializing event in protein aggregation is thought to be the formation of an abnormal oligomer. For instance, ␤-amyloid and ␣-synuclein undergo conformational changes in Alzheimer disea...

1 The mediators of nonadrenergic, noncholinergic (NANC) inhibitory responses in longitudinal muscle of rat distal colon were studied.2 An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptors, PACAP638, concentration-dependently inhibited the rapid relaxation of the longitudinal muscle induced by electrical field stimulation (EFS), resulting in a maximal inhibition of 47% at 3 MM.3 PACAP638 inhibited the relaxation by 75% in the presence of the vasoactive intestinal peptide (VIP) receptor antagonist, VIPIO 28 at 3 Mm, which inhibited the relaxation by 44%.4 An antagonist of large conductance Ca2+-activated K+ channels, charybdotoxin, concentrationdependently inhibited the rapid relaxation of the longitudinal muscle, resulting in a maximal inhibition of 58% at 100 nM.5 An antagonist of small conductance Ca2 +-activated K+ channels, apamin, concentration-dependently inhibited the relaxation (58% at 1 gM).6 Treatment with both K+ channel antagonists resulted in 84% inhibition of the EFS-induced relaxation, which is comparable to the extent of inhibition induced by PACAP638 plus VIPI0O28. 7 The inhibitory effect of VIP1028 and of apamin, but not of charybdotoxin was additive: the same applied to PACAP6-38 and charybdotoxin, but not apamin. 8 Exogenously added VIP (100 nM-1 Mm) induced a slow gradual relaxation of the longitudinal muscle. Charybdotoxin, but not apamin significantly inhibited the VIP-induced relaxation. VIPI0O28, but not PACAP638 selectively inhibited the VIP-induced relaxation.9 Exogenously added PACAP (10-100 nM) also induced slow relaxation. Apamin and to a lesser extent, charybdotoxin, inhibited the PACAP-induced relaxation. PACAP638, but not VIPI0O28 selectively inhibited the PACAP-induced relaxation.10 Apamin at 100 nM inhibited inhibitory junction potentials (ij.ps) induced by a single pulse of EFS. Apamin also inhibited a rapid phase, but not a delayed phase of ij.ps induced by two pulses at 10 Hz. VIPIO28 did not inhibit i.j.ps induced by a single pulse, but significantly inhibited the delayed phase at two pulses. A combination of apamin and VIPIO28 abolished the ij.ps induced by two pulses.11 Both VIP and PACAP induced slow hyperpolarization of the cell membrane of the longitudinal muscle. Apamin inhibited the PACAP-, but not VIP-induced hyperpolarization. 12 From these findings it is suggested that VIP and PACAP are involved in NANC inhibitory responses of longitudinal muscle of the rat distal colon via activation of charybdotoxin-and apaminsensitive K+ channels, respectively.

In the present study, we examined the expression and the localization of apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK channels) in the mouse intestine. SK3-immunoreactivity (IR) was detected in both ileum and colon. Double staining experiments showed that SK3-IR was colocalized with prolyl 4-hydroxylase (PH(alpha))-IR, but not with c-Kit-IR which are markers of fibroblast cells and the interstitial cells of Cajal (ICC), respectively. Although SK3-IR was colocalized with vimentin-IR, which is another marker of ICC, the reactivity of SK3-immunopositive cells was weaker than that of ICC. The SK3-immunopositive cells were similarly present in the intestine of c-Kit mutant mice (W/W(V)), in which ICC were absent, and its wild-type mice. The immuno-electron microscopic analysis indicated that SK3 was localized in the cells that had some similar morphological features to ICC, but obviously different from ICC. The SK3-immunopositive cells had gap junctions with the smooth muscle cells. The gap junctions were smaller than those between ICC and smooth muscle cells. These results indicate expression of SK3 in fibroblast-like cells, but not in ICC, and suggest participation of the cells in the intestinal motility.

The mediators of non‐adrenergic non‐cholinergic (NANC) relaxation of the longitudinal muscle of rat proximal, middle and distal colon were examined in vitro. Electrical transmural stimulation (TMS) of proximal, middle and distal segments of rat colon induced NANC relaxations which were inhibited by tetrodotoxin (1 μm), but not by atropine (1 μm) or guanethidine (4 μm). In the proximal colon, l‐nitro‐arginine (N5‐nitroamidino‐l‐2,5‐diaminopentanoic acid) inhibited the TMS‐induced NANC relaxation and l‐arginine (1 mm) reversed this inhibition. Nitric oxide (0.3–10 μm) induced relaxation of the proximal segment. NANC relaxation of the proximal segments was still evident after desensitization to vasoactive intestinal peptide (VIP). A VIP antagonist (VIP 10–28, 10 μm) had no effect on the TMS‐induced NANC relaxation, which was also resistant to α‐chymotrypsin (2 units ml−1) and a substance P antagonist ([d‐Pro2, d‐Trp7,9]substance P, 1 μm). In the middle colon, l‐nitro‐arginine did not inhibit the TMS‐induced NANC relaxation in 6 of 9 preparations tested and partially inhibited the relaxation in the other 3 preparations. l‐Arginine did not reverse the partial inhibition. Complete desensitization to VIP was not achieved in the middle colon. The VIP antagonist had no effect on the TMS‐induced NANC relaxation. After α‐chymotrypsin treatment of the segment, desensitization of the segments to substance P, or in the presence of the substance P antagonist, the TMS‐induced NANC relaxation was augmented. In the distal colon, l‐nitro‐arginine did not have any significant effect on the TMS‐induced relaxation and nitric oxide did not induce relaxation. The VIP antagonist significantly inhibited TMS‐induced NANC relaxation. α‐Chymotrypsin‐treatment of the distal segments resulted in significant inhibition of NANC relaxation. No desensitization to substance P was achieved. Treatment with the substance P antagonist had no effect. These results suggest that nitric oxide is the mediator of the NANC inhibitory response in the proximal region of rat colon; in the middle colon, substance P acts as an excitatory neurotransmitter, antagonizing the NANC relaxation caused by the mediator of the response, which is still uncertain. Our results also suggest that VIP is the most likely candidate as a NANC transmitter in the distal colon.

Hydrochloric acid (HCl) is produced in parietal cells of gastric epithelium by a H+‐K+ pump. Protons are secreted into the gastric lumen in exchange for K+ by the action of the H+‐K+‐ATPase. Luminal K+ is essential for the operation of the pump and is thought to be supplied by unidentified K+ channels localized at the apical membrane of parietal cells. In this study, we showed that histamine‐ and carbachol‐induced acid secretion from isolated parietal cells monitored by intracellular accumulation of aminopyrine was depressed by Ba2+, an inhibitor of inwardly rectifying K+ channels. Among members of the inwardly rectifying K+ channel family, we found with reverse transcriptase‐polymerase chain reaction analyses that Kir4.1, Kir4.2 and Kir7.1 were expressed in rat gastric mucosa. With immunohistochemical analyses, Kir4.1 was found to be expressed in gastric parietal cells and localized specifically at their apical membrane. The current flowing through Kir4.1 channel expressed in HEK293T cells was not affected by reduction of extracellular pH from 7.4 to 3. These results suggest that Kir4.1 may be involved in the K+ recycling pathway in the apical membrane which is required for activation of the H+‐K+ pump in gastric parietal cells.

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We investigated the subtype of presynaptic muscarinic receptors associated with inhibition of acetylcholine (ACh) release in the mouse small intestine. We measured endogenous ACh released from longitudinal muscle with myenteric plexus (LMMP) preparations obtained from M1-M5 receptor knockout (KO) mice. Electrical field stimulation (EFS) increased ACh release in all LMMP preparations obtained from M1-M5 receptor single KO mice. The amounts of ACh released in all preparations were equal to that in the wild-type mice. Atropine further increased EFS-induced ACh release in the wild-type mice. Unexpectedly, atropine also increased, to a similar extent, EFS-induced ACh release to the wild-type mice in all M1-M5 receptor single KO mice. In M2 and M4 receptor double KO mice, the amount of EFS-induced ACh release was equivalent to an atropine-evoked level in the wild-type mouse, and further addition of atropine had no effect. M2 receptor immunoreactivity was located in both smooth muscle cells and enteric neurons. M4 receptor immunoreactivity was located in the enteric neurons, being in co-localization with M2 receptor immunoreactivity. These results indicate that both M2 and M4 receptors mediate the muscarinic autoinhibition in ACh release in the LMMP preparation of the mouse ileum, and loss of one of these subtypes can be compensated functionally by a receptor that remained. M1, M3, and M5 receptors do not seem to be involved in this mechanism.

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