Ethanolamine Plasmalogen Suppresses Apoptosis in Human Intestinal Tract Cells in Vitro by Attenuating Induced Inflammatory Stress.

Ethanolamine plasmalogen (PlsEtn) is a subtype of ethanolamine glycerophospholipids (EtnGpl). Recently, PlsEtn has attracted increasing research interest due to its beneficial effects in health and disease; however, its functional role in colonic health has not been well established. This study was conducted to determine the mechanism underlying the antiapoptotic effect of PlsEtn in human intestinal tract cells under induced inflammatory stress. Lipopolysaccharide induced apoptosis of differentiated Caco-2 cells, which was suppressed by EtnGpl in a dose-dependent manner. Cells treated with ascidian muscle EtnGpl containing high levels of PlsEtn demonstrated a lower degree of apoptosis, and downregulated TNF-α and apoptosis-related proteins compared to those treated with porcine liver EtnGpl containing low PlsEtn. This indicates that PlsEtn exerted the observed effects, which provided protection against induced inflammatory stress. Overall, our results suggest that PlsEtn with abundant vinyl ether linkages is potentially beneficial in preventing the initiation of inflammatory bowel disease and colon cancer.

C-terminal region regulates the functional expression of human noradrenaline transporter splice variants.

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl--dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

Nonsteroidal anti-inflammatory drugs potentiate 1-methyl-4-phenylpyridinium (MPP+)-induced cell death by promoting the intracellular accumulation of MPP+ in PC12 cells.

In this study, we investigated the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on 1-methyl-4-phenylpyridinium (MPP(+))-induced cell death in PC12 cells. Coincubation of PC12 cells with indomethacin, ibuprofen, ketoprofen, or diclofenac, but not aspirin or N-[2-(cyclohexyloxy)-4-nitrophenyl]methanosulfonamide (NS-398), significantly potentiated the MPP(+)-induced cell death. In contrast, these NSAIDs had no effect on rotenone-induced cell death. The potentiating actions of these NSAIDs were not suppressed by treatment with phenyl-N-butyl-nitrone, a radical scavenger; N-acetyl-l-cysteine, an antioxidant; Ac-DEVD-CHO, a selective caspase-3 inhibitor; or 2-chloro-5-nitro-N-phenylbenzamide (GW9662), a selective antagonist of peroxisome proliferator-activated receptor gamma. Furthermore, we observed that DNA fragmentation, which is one of the hallmarks of apoptosis, was not induced by coincubation with MPP(+) and NSAIDs. We confirmed that coincubation of PC12 cells with 30 microM MPP(+) and 100 microM indomethacin, ibuprofen, ketoprofen, or diclofenac led to a significant increase in the accumulation of intracellular MPP(+) compared with incubation with 30 microM MPP(+) alone. In addition, these NSAIDs markedly reduced the efflux of MPP(+) from PC12 cells. (3-(3-(2-(7-Chloro-2-quinolinyl) ethenyl) phenyl ((3-dimethyl amino-3oxo-propyl) thio) methyl) propanoic acid (MK 571), which is an inhibitor of multidrug resistance proteins (MRPs), mimicked the NSAIDs-induced effects, increasing cell toxicity and promoting the accumulation of MPP(+). Moreover, some types of MRPs' mRNA were detected in PC12 cells. These results suggest that some NSAIDs might cause a significant increase in the intracellular accumulation of MPP(+) via the suppression of reverse transport by the blockade of MRP, resulting in the potentiation of MPP(+)-induced cell death.

Regulation of dopamine and MPP+ transport by catecholamine transporters.

Following exocytotic release of the biogenic amine neurotransmitters, norepinephrine and dopamine, are removed from the synaptic cleft by the respective transporter, norepinephrine transporter (NET) and dopamine transporter (DAT) located on the plasma membrane. The catecholamine transporters are the molecular targets for psychoactive drugs as well as drugs of abuse such as cocaine and amphetamine and the Parkinsonism-inducing neurotoxin, MPP+. Nicotine regulates the transport of catecholamines and MPP+ and may exert self-medicating effects for depression, schizophrenia and attention deficit hyperactivity disorder, and neuroprotective effects against MPP+ through the regulation of the transporters. The availability of cDNAs of these transporters has permitted detailed pharmacological studies in heterologous expression systems for determining the mechanisms of action of nicotine on transporters. Moreover, functional analysis of the effect of single amino acid substitution suggests that specific residues in DAT molecules may play a significant role in interaction with MPP+ and cocaine, and thus would promise a development of novel drugs with diverse chemical structures.

Chronic inhibition of the norepinephrine transporter in the brain participates in seizure sensitization to cocaine and local anesthetics.

The involvement of chronic inhibition of monoamine transporters (MAT) in the brain with respect to sensitization to cocaine- and local anesthetic-induced seizures was studied in mice. Repeated administration of subconvulsive doses of meprylcaine as well as cocaine, both of which inhibit MAT, but not lidocaine, which does not inhibit MAT, increased seizure activity and produced sensitization to other local anesthetics. The effects of five daily treatments of monoamine transporter inhibitors on lidocaine-induced convulsions were examined 2 or 3 days after the last dose of the inhibitors. Daily treatments of GBR 12935, a specific inhibitor of dopamine uptake, significantly increased the incidence and the intensity of lidocaine-induced convulsions at 20 mg/kg and decreased the threshold of the convulsions. Daily treatments of desipramine and maprotiline, selective norepinephrine uptake inhibitors, markedly increased the incidence and intensity of lidocaine-induced convulsions, and decreased the threshold in a dose-dependent manner at between 5 and 20 mg/kg. Daily treatments of citalopram, a selective serotonin uptake inhibitor, at 10 and 20 mg/kg, produced no significant increase in the incidence or intensity of lidocaine-induced convulsions, but decreased the threshold of the convulsions. These results suggest that the chronic intermittent inhibition of monoamine uptake increases susceptibility to cocaine- and local anesthetic-induced seizures, and the norepinephrine transporter is an integral component of this sensitization.

Pituitary adenylate cyclase-activating polypeptide induces a sustained increase in intracellular free Ca(2+) concentration and catechol amine release by activating Ca(2+) influx via receptor-stimulated Ca(2+) entry, independent of store-operated Ca(2+) channels, and voltage-dependent Ca(2+) channels in bovine adrenal medullary chromaffin cells.

Characteristics of pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increase of Ca(2+) entry and catecholamine (CA) release were studied in bovine adrenal medullary chromaffin cells. PACAP induced intracellular free Ca(2+) concentration ([Ca(2+)](i)), showing an initial transient [Ca(2+)](i) rise followed by a sustained rise and CA release, which were not blocked by the blocking agents for nicotinic acetylcholine receptor (nAChR) channel, the voltage-dependent Ca(2+) channel (VOC), or the Na(+) channel. The sarcoendoplasmic Ca(2+)-ATPase inhibitors thapsigargin and cyclopiazonic acid did not affect the PACAP-induced sustained rise of [Ca(2+)](i), but did inhibit the initial [Ca(2+)](i) rise. In cells pretreated with cyclopiazonic acid or membrane-permeable, low-affinity Ca(2+) chelator N',N',N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, PACAP further stimulated the entry of Ca(2+) or Mn(2+), whereas these treatments masked [Ca(2+)](i) dynamics induced by bradykinin. PACAP-induced sustained [Ca(2+)](i) rise and Mn(2+) entry were enhanced by acidic extracellular solution and reduced by alkalinization, whereas thapsigargin-induced Mn(2+) entry was regulated by the opposite. PACAP-induced [Ca(2+)](i) rise and Mn(2+) entry were not affected by blockers of cAMP-dependent protein kinase, phospholipase C, or protein kinase C. All store-operated Ca(2+) channel (SOC) blocking agents tested inhibited thapsigargin-induced Mn(2+) entry. 1(beta-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenylethyl)-1H-imidazole hydrochloride (SK&F 96365), (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide, and econazole inhibited PACAP-induced Ca(2+) or Mn(2+) entry, whereas GdCl(3), 7,8-benzoflavone, nor-dihydroguaiaretic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, fulfenamic acid, and niflumic acid did not. SK&F 96365 and econazole but not GdCl(3) inhibited PACAP-induced CA release. These results suggest that PACAP activates a novel Ca(2+) entry pathway associated with sustained CA release independent of the nAChR channel, VOC and SOC, activated by acid pH, with different sensitivity to blockers of SOC. This pathway may provide a useful model for the study of receptor-operated Ca(2+) entry.

Identification and functional characterization of the novel isoforms of bovine norepinephrine transporter produced by alternative splicing.

Analyzing variation of bovine norepinephrine transporter (NET) at the 3'-region by RT-PCR in the adrenal glands and the brain revealed four isoforms of NET produced by alternative splicing of four cassettes (C0, C1, C2 and C3) encoded by exons 12-15, designated bNET1a (C0-C1-C2, formerly designated bNET1), bNET1b (C0-C2), bNET2a (C0-C1-C3) and bNET2b (C0-C3, formerly designated bNET2), respectively. Expression of these isoforms in COS-7 cells revealed that the isoforms that contain the C1 cassette encoded by exon 13 (bNET1a and bNET2a) showed a significant increase in [(3)H]norepinephrine uptake and [(3)H]nisoxetine binding, whereas the isoforms which lack the C1 cassette (bNET1b and bNET2b) failed to display those activities despite the selection of either exon 14 or exon 15. These results suggest that the region encoded by exon 13 is indispensable for NET functional expression.

Interleukin-1beta-induced substance P release from rat cultured primary afferent neurons driven by two phospholipase A2 enzymes: secretory type IIA and cytosolic type IV.

We previously described that recombinant interleukin-1beta (IL-1beta) induced the significant release of substance P (SP) via a cyclooxygenase (COX) pathway in primary cultured rat dorsal root ganglion (DRG) cells. In the present study, we examined the involvement of two types of phospholipase A2 (PLA2) enzymes, which lie upstream of COX in the prostanoid-generating pathway, in the IL-1beta-induced release of SP from DRG cells. The expression of type IIA secretory PLA2 (sPLA2 -IIA) mRNA was undetectable by ribonuclease protection assay in non-treated DRG cells, while in DRG cells incubated with 1 ng/mL of IL-1beta, the expression was induced in a time-dependent manner. On the other hand, type IV cytosolic PLA2 (cPLA2 ) mRNA was constitutively expressed in the non-treated DRG cells, and treatment with 1 ng/mL of IL-1beta for 3 h significantly increased the levels of cPLA2 mRNA. The IL-1beta-induced SP release was significantly inhibited by the sPLA2 inhibitor, thioetheramide phosphorylcholine (TEA-PC), and the cPLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3 ). Furthermore AACOCF3 suppressed the induction of sPLA2 -IIA mRNA expression induced by IL-1beta. These observations suggested that two types of PLA2, sPLA2 -IIA and cPLA2, were involved in the IL-1beta-induced release of SP from DRG cells, and that the functional cross-talk between the two enzymes might help to control their activity in the prostanoid-generating system in DRG cells. These events might be key steps in the inflammation-induced hyperactivity in primary afferent neurons of spinal cord.