The Nitrated Form of Nateglinide Induces Apoptosis in Human Pancreatic Cancer Cells Through a Caspase-dependent Mechanism.

BACKGROUND/AIM: Nitric oxide (NO) has antitumor activity against various solid tumor cell types in addition to its vasodilatory effect. In the current study, we investigated the effect and mechanism of the synthetic nitrated form (NO2-NAT) of nateglinide, a hypoglycemic agent, on the induction of cell death in human pancreatic cancer cells. MATERIALS AND METHODS: NO production was evaluated by measuring nitrite (NO2) and nitrate (NO3) (NOx). Apoptotic cell numbers were determined using annexin V. RESULTS: NO2-NAT released nitrate and nitrite ions immediately upon dissolving in aqueous solution. NO2-NAT caused significant extracellular leakage of lactate dehydrogenase (LDH) in the human pancreatic cancer cell lines AsPC1 and BxPC3, and increased annexin-positive cells in a time- and concentration-dependent manner. NO2-NAT also significantly increased the activity of caspases 3 and 7. Exposure to Z-VAD-FMK, a caspase inhibitor, significantly suppressed NO2-NAT-induced cell death. CONCLUSION: These results indicated that NO2-NAT induces apoptosis in human pancreatic cancer cells and may serve as a new NO-based chemotherapeutic agent for the treatment of pancreatic cancer.

Synthesis and biological activities of novel structural analogues of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand.

Novel analogues of 2-arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand, were developed. Chemical synthesis of these analogues (2-AGA105 and 2-AGA109) was accomplished starting from 2-octyn-1-ol and diethyl malonate and employing Wittig coupling of triene phosphonate with an aldehyde intermediate in a convergent and stereoselective manner. These analogues should be useful lead compounds for the development of novel 2-AG mimetics.

Generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in picrotoxinin-administered rat brain.

The levels of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, and other molecular species of monoacylglycerols in rat brain were examined. In this study, we sacrificed the animals in liquid nitrogen to minimize postmortem changes. We found that rat brain contains 0.23 nmol/g tissue of 2-arachidonoylglycerol, which accounts for 10.5% of the total monoacylglycerol present in this tissue. We next investigated the level of 2-arachidonoylglycerol after in vivo stimulation with picrotoxinin. We found that the level of 2-arachidonoylglycerol was elevated markedly in picrotoxinin-administered rat brain (4- to 6-fold over the control level). Changes in the levels of other molecular species were relatively small or negligible. Several cannabimimetic molecules as well as Delta(9)-tetrahydrocannabinol are known to depress neurotransmission and to exert anticonvulsant activities; endogenous 2-arachidonoylglycerol produced during neural excitation may play a regulatory role in calming the enhanced synaptic transmission.

Evidence that the cannabinoid CB1 receptor is a 2-arachidonoylglycerol receptor. Structure-activity relationship of 2-arachidonoylglycerol, ether-linked analogues, and related compounds.

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca2+ concentrations in NG108-15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.

Is the cannabinoid CB1 receptor a 2-arachidonoylglycerol receptor? Structural requirements for triggering a Ca2+ transient in NG108-15 cells.

The effects of delta9-tetrahydrocannabinol and 2-arachidonoylglycerol on the intracellular free Ca2+ concentration ([Ca2+]i) in NG108-15 cells were examined in detail. We found that delta9-tetrahydrocannabinol induces a rapid, modest increase in [Ca2+]i. The response was detectable with 3 nM delta9-tetrahydrocannabinol. We also found that very low concentrations of 2-arachidonoylglycerol elicit a rapid, more prominent increase in [Ca2+]i. Such a response was observed not only in NG108-15 cells but also in N18TG2 cells. The response induced by 2-arachidonoylglycerol in either NG108-15 cells or N18TG2 cells was abolished by pretreatment of the cells with a cannabinoid CB1 receptor specific antagonist, SR141716A, suggesting that 2-arachidonoylglycerol interacts with the CB1 receptor to induce the response. The results of an experiment involving a phospholipase C inhibitor suggested that phospholipase C is involved in the rapid increase in [Ca2+]i induced by 2-arachidonoylglycerol. We also found that 1(3)-arachidonoylglycerol exhibits similar activity to that of 2-arachidonoylglycerol, although its activity at low concentrations was somewhat weak compared with that of 2-arachidonoylglycerol. We further confirmed that several structural analogues of 2-arachidonoylglycerol were less active compared with 2-arachidonoylglycerol. These results suggest that the structure of 2-arachidonoylglycerol is strictly recognized by the CB1 receptor, which raises the possibility that the CB1 receptor is originally a 2-arachidonoylglycerol receptor.

Enzymatic synthesis of oleamide (cis-9, 10-octadecenoamide), an endogenous sleep-inducing lipid, by rat brain microsomes.

The enzymatic synthesis of a novel sleep-inducing lipid, oleamide (cis-9,10-octadecenoamide), was studied using rat brain subcellular fractions as enzyme sources. We found that oleamide was formed from oleic acid and ammonia on incubation with a brain homogenate. The enzyme activity catalyzing the formation of oleamide from oleic acid and ammonia was highest in the microsomal fraction among the subcellular fractions. Boiled microsomes did not exhibit appreciable enzyme activity. These results strongly suggest that oleamide can be synthesized enzymatically in the brains of stimulated animals.

Transacylase-mediated and phosphodiesterase-mediated synthesis of N-arachidonoylethanolamine, an endogenous cannabinoid-receptor ligand, in rat brain microsomes. Comparison with synthesis from free arachidonic acid and ethanolamine.

The levels of N-arachidonoylethanolamine (anandamide), an endogenous cannabinoid-receptor ligand, and a relevant molecule, N-arachidonoylphosphatidylethanolamine (N-arachidonoylPtdEtn), in rat brain were investigated using a newly developed sensitive analytical method. We found that rat brain contains small but significant amounts of these two types of N-arachidonoyl lipids (4.3 pmol/g tissue and 50.2 pmol/g tissue, respectively). Then, we investigated how N-arachidonoylethanolamine (anandamide) is produced in the brain. We found that anandamide can be formed enzymatically via two separate synthetic pathways in the brain: enzymatic condensation of free arachidonic acid and ethanolamine; and formation of N-arachidonoylPtdEtn from PtdEtn and arachidonic acid esterified at the 1-position of phosphatidyl-choline (PtdCho), and subsequent release of anandamide from N-arachidonoylPtdEtn through the action of a phosphodiesterase. We confirmed that rat brain contains both the enzyme activities and lipid substrates involved in these reactions. Several lines of evidence strongly suggest that the second pathway, rather than the first one, meets the requirements and conditions for the synthesis of various species of N-acylethanolamine including anandamide in the brain.

Inositol 1,4,5-trisphosphate accumulation in brain of lithium-treated rats.

The mechanism of action of lithium as a drug for patients with affective disorders was investigated. Three-week-old male rats were orally administered 2.7 mEq Li2CO3/kg/d for 1 or 3 wk, and phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), inositol phosphate (IP), inositol diphosphate (IP2) and inositol triphosphate (IP3) levels in brain were measured. The levels of IP were increased 1.7 and 2.4 times after 1 wk and 3 wk of lithium administration, respectively, while PI, PIP, PIP2, IP2 and IP3 levels were not altered. IP3 was further fractionated by high-performance liquid chromatography into I-1,3,4-P3 and I-1,4,5-P3. In the control rat brain, the relative percentages of I-1,3,4-P3 and I-1,4,5-P3 were 95.8 and 4.2, respectively. However, after 3 wk of lithium administration, the values were changed to 69.6 and 30.3%, respectively. This increase in the neurotransducer I-1,4,5-P3 in the brain may be relevant to the mechanism of action in the lithium treatment of patients with manic-depressive disorders.

Turnover rates of the molecular species of ethanolamine plasmalogen of rat brain.

1,2-Diradyl-3-acetylglycerols prepared from 1-O-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (alkenylacyl-GPE, ethanolamine plasmalogen) and 1-alkyl-2-acylglycero-3-phosphoethanolamine (alkylacyl-GPE) of rat brain at 18 days of age were subfractionated into six species by AgNO3-impregnated TLC. The percent compositions of substractions were compared with that of 1,2-diacylglycero-3-phosphoethanolamine (diacyl-GPE). The incorporation rate of [1-3H]glycerol into each molecular species was also estimated to examine the turnover rate and selective synthesis of molecular species of ethanolamine phosphoglycerides (EPG). Among the molecular species of EPG, a major proportion contained polyunsaturated fatty chains, and the sum of tetraene-, pentaene-, and hexaene-containing species was greater than 65% in common with three classes of EPG. It was possible to calculate the turnover time, synthesis rate, and synthesis rate constant of ethanolamine plasmalogen in myelinating rat brain by the equation of Zilversmit et al. since the time-dependent change of specific activity and the distribution of molecular species indicated that each molecular species of alkenylacyl-GPE is synthesized from the corresponding species of alkylacyl-GPE. The observed turnover time of ethanolamine plasmalogen was about 5 h. The observed turnover times of the various molecular species were of the order: tetraene greater than or equal to hexaene greater than pentaene greater than or equal to monoene greater than or equal to diene. The synthesis rate constants of each molecular species, in the formation of alkenylacyl-GPE from alkylacyl-GPE, were of the order: hexaene greater than tetraene greater than pentaene greater than diene greater than or equal to monoene.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective incorporation of docosahexaenoic acid in rat brain.

The incorporation of [14C]docosahexaenoic acid (22:6) into various lipid classes was compared with that of [3H]arachidonic acid (20:4) in developing rat brains in order to characterize the retention of 22:6. The percentage distribution of 20:4 in neutral glycerides was similar to the mass distribution and, of the phospholipids, 20:4 was incorporated into choline glycerophospholipids and inositol glycerophospholipids in relatively higher amounts. The incorporation of labeled 22:6 differed from that of 20:4, as it was incorporated into ethanolamine glycerophospholipids and triacylglycerols in greater amounts in comparison with its mass distribution. Further division of ethanolamine glycerophospholipids into diacyl, alkylacyl and alkenylacyl types revealed that the high incorporation of radioactive 22:6 in ethanolamine glycerophospholipids was predominantly due to its rapid incorporation into diacyl and alkylacyl compounds. The distributions of both radioactively labeled 20:4 and 22:6 among the three types of ethanolamine glycerophospholipids were compared with those of radioactively labeled glycerol. The value of [3H]20:4/[3H]glycerol was much higher in alkenylacyl-GPE than in alkylacyl-GPE, and this indicates that 20:4 is incorporated into alkenylacyl-GPE by a deacylation-reacylation mechanism. In addition, [14C]22:6 was incorporated into ether-linked ethanolamine phospholipids in a similar manner to 20:4. This observation suggests that 22:6 may be incorporated into alkenylacyl-GPE by the same rearranging system, while another possibility is that preferential synthesis of hexaene species may occur in the desaturation of alkylacyl-GPE.

Changes in the composition of fatty chains of diacyl, alkylacyl and alkenylacyl ethanolamine and choline phosphoglycerides during the development of chick heart ventricular cells. High accumulation of 22-carbon fatty acid in ether phospholipids.

The phospholipids of embryonic chick ventricular cells were analysed at various developmental stages with respect to the composition of alkylacyl, alkenylacyl and diacyl ethanolamine and choline phosphoglycerides and for the fatty chain composition of these lipid classes. The percentage of alkylacyl and alkenylacyl choline phosphoglycerides increased in the course of the development. The fatty chain composition of ether-linked phosphoglycerides was significantly different from that of the diacyl compound. In general, both ether choline and ethanolamine phosphoglycerides consisted of a significantly higher percentage of 22-carbon fatty chains, such as 22:4, 22:5 and 22:6, compared to that of the diacyl compounds, throughout the earlier stage of development. During development, there was a dramatic increase of 20:4 and a decrease of 22:6, mainly in total ethanolamine phosphoglycerides but also in choline phosphoglycerides. A particularly significant decrease of 22:6 was found with diacyl ethanolamine phosphoglycerides. When 14C-labeled 22:4 and 3H-labeled 20:4 were incorporated into the ethanolamine and choline phosphoglycerides of ventricles in vitro, it was observed that 22:4 was mainly associated with ether phospholipids, especially the ethanolamine alkyl ether phospholipids, suggesting that there is a high selectivity of 22-carbon fatty acid to ether phospholipids during the synthesis of these compounds.

Electrical membrane phenomena in spherules from proteinoid and lecithin.

Spontaneous and induced electrical phenomena resembling membrane and action potentials in natural excitable cells have been observed in artificial cells. These artificial cells were made from thermal proteinoid and lecithin in a solution of potassium acid phosphate with glycerol.

Excitability of crayfish giant axons in sodium-free external media containing hydrazinium, hydroxylamine, or guanidinium ions.

Giant axons of crayfish were excitable in sodium-free external media containing hydrazinium, hydroxylamine, or guanidinium ions. Action potentials in these solutions were slightly smaller in size and a little longer in duration than those in standard Harreveld's solution. The resting potential was not affected in hydrazine or in hydroxylamine saline, but decreased in gaunidine saline. The membrane potential at the peak of the spike (V) was plotted against the univalent cation concentrations in the external medium. The relationship could be expressed by an equation: V = VO + (RT/F) ln ([Na] + alpha[C]), where VO and alpha are constants and [Na] and [C] are concentrations of sodium ions and the tested cations, respectively. The parameter alpha was considered as an indicator to express the ability of substitution of the cation for sodium ion. The order of the ability of the substitution was Hydroxylamine greater than Hydrazinium greater than Guanidinium greater than Choline.