4-Hydroxy-2-Nonenal Levels Increase in the Plasma of Patients with Adult Respiratory Distress Syndrome as Linoleic Acid Appears to Fall

Gas chromatography-mass spectrometry has been applied to the analysis of plasma linoleic acid and one of its oxidation products, 4-hydroxy-2-nonenal (HNE), in adult patients with the acute respiratory distress syndrome (ARDS). Peak areas of total ion chromatograms showed there to be negative correlations between loss of linoleic acid and formation of HNE (measured by selective ion monitoring) in 7 out 10 patients studied. When HNE was quantitated by selective ion monitoring, with reference to a pure standard of HNE and an internal standard of nonanoic acid, ARDS patients showed significantly increased levels of HNE (0.412 ± 0.023 nmol/ml) compared with normal healthy controls (0.205 ± 0.018 nmol/ml).     

Aluminum alters calcium transport in plasma membrane and endoplasmic reticulum from rat brain

Calcium is actively transported into intracellular organelles and out of the cytoplasm by Ca²⁺/Mg²⁺-ATPases located in the endoplasmic reticulum and plasma membranes. We studied the effects of aluminum on calcium transport in the adult rat brain. We examined ⁴⁵Ca-uptake in microsomes and Ca²⁺-ATPase activity in microsomes and synaptosomes isolated from the frontal cortex and cerebellum of adult male Long-Evans rats. ATP-dependent⁴⁵Ca-uptake was similar in microsomes from both brain regions. The addition of 50-800 μM AICI₃ resulted in a concentration-dependent inhibition of ⁴⁵Ca-uptake. Mg²⁺-dependent Ca²⁺-ATPase activity was significantly lower in synaptosomes compared to microsomes in both frontal cortex and cerebellum. In contrast to the uptake studies, AICI³ stimulated Mg²⁺-dependent Ca²⁺-ATPase activity in both microsomes and synaptosomes from both brain regions. To determine the relationship between aluminum and Mg²⁺, we measured ATPase activity in the presence of increasing concentrations of Mg²⁺ or AICI³. Maximal ATPase activity was obtained between 3 and 6 mM Mg²⁺. When we substituted AICI³ for Mg²⁺, ATPase activity was also stimulated in a concentration-dependent manner, but to a greater extent than with Mg²⁺. One interpretation of these data is that aluminum acts at multiple sites to displace both Mg²⁺ and Ca²⁺, increasing the activity of the Ca²⁺-ATPase, but disrupting transport of calcium.     

Effects of hypothalamic microinjection of PGE2 on body temperature and sympathetic nervous activities in the rabbit

The febrile response and sympathetic nervous response to hypothalamic microinjections of prostaglandin E₂ (PGE₂) were investigated in anesthetized rabbits. Microninjection of PGE₂ (500–1000 ng) caused an increase in rectal temperature of more than 0.3°C in 13 of 50 loci in the preoptic and anterior hypothalamic area (PO/AH). At 8 of these 13 loci, PGE₂ elicited response patterns in the sympathetic nervous system, such as an increase in cutaneous sympathetic nervous activity and decrease in renal sympathetic nervous activity. This pattern of sympathetic nervous responses was induced with a simultaneous increase in rectal temperature of more than 0.5°C. The 8 loci were distributed in the preoptic area, especially in the vicinity of the supraoptic nucleus. Electrolytic lesions of this region were made bilaterally, and intracerebroventricular injection of PGE₂ (8 µg/kg) was found to inhibit fever and sympathetic activity. The results demonstrate that the action of PGE₂ is responsible for the response patterns of sympathetic twigs during fever. The preoptic area, especially in the vicinity of the supraoptic nucleus, is most sensitive to PGE₂ for the patternized response of sympathetic neurons and fever.     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Regulation of taurine transport in Ehrlich ascites tumor cells

Summary Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized. Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na, 1Cl, 1taurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the leukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A₂ is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD₄. The swelling-induced taurine efflux is also inhibited by addition of the 5-lipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-lipoxygenase pathway. LTD₄ seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the Cl^– channels, i.e., via a depolarization of the cell membrane.     

A Polymerase Chain Reaction Method for Identification of Five Major Meloidogyne Species

A polymerase chain reaction (PCR) method for discriminating Meloidogyne incognita, M. arenaria, M. javanica, M. hapla, and M. chitwoodi was developed. Single juveniles were ruptured in a drop of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3'' portion of the mitochondrial gene coding for cytochrome oxidase subunit II and in the 16S rRNA gene. Following PCR amplification, fragments of three sizes were detected. The M. incognita and M. javanica reactions produced a 1.7-kb fragment; the M. arenaria reaction, a 1.1-kb fragment; and the M. hapla and M. chitwoodi reactions resulted in a 0.52-kb fragment. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Dra I digestions of the 0.52-kb amplification product produced a characteristic three-banded pattern in M. chitwoodi, versus a two-banded pattern in M. hapla. Hinf I digestion of the 1.7-kb fragment produced a two-banded pattern in M. javanica, versus a three-banded pattern in M. incognita. Amplification and digestion of DNA from juveniles from single isolates of M. marylandi, M. naasi, and M. nataliei indicated that the diagnostic application of this primer set may extend to less frequently encountered Meloidogyne species.     

Ammonium Injection Induces an N-Methyl-d-Aspartate Receptor-Mediated Proteolysis of the Microtubule-Associated Protein MAP-2

Abstract: We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule-associated protein 2 (MAP-2). Activation of the N -methyl- d -aspartate (NMDA) receptor dephosphorylates MAP-2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP-2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP-2. Also, the amount of MAP-2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP-2 was decreased by ∼75% 1-2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP-2 was prevented completely by injection of 2 mg/kg MK-801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor. l -Carnitine, which protects rats against ammonia toxicity, also prevented MAP-2 degradation. Because activation of the NMDA receptor increases [Ca²⁺]_i, we determined whether rat brain contains a Ca²⁺-dependent protease that selectively degrades MAP-2. We show that there is a cytosolic Ca²⁺-dependent protease that degrades MAP-2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca²⁺]_i, which activates calpain I. This, in turn, selectively degrades MAP-2. Possible implications in chronic hyperammonemic states and in the mechanism of ammonia toxicity are discussed.     

Phospholipase A2 Modulates Different Subtypes of Excitatory Amino Acid Receptors: Autoradiographic Evidence

Abstract: Exogenous phospholipases have been used extensively as tools to study the role of membrane lipids in receptor mechanisms. We used in vitro quantitative autoradiography to evaluate the effect of phospholipase A₂ (PLA₂) on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat brain. PLA₂ pretreatment induced a significant increase in α-[³H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([³H]AMPA) binding in the stratum radiatum of the CA1 region of the hippocampus and in the stratum moleculare of the cerebellum. No modification of [³H]AMPA binding was found in the stratum pyramidale of the hippocampus at different ligand concentrations. [³H]-Glutamate binding to the metabotropic glutamate receptor and the non-NMDA-, non-kainate-, non-quisqualate-sensitive [³H]glutamate binding site were also increased by PLA₂ pretreatment. [³H]Kainate binding and NMDA-sensitive [³H]glutamate binding were minimally affected by the enzyme pretreatment. The PLA₂ effect was reversed by EGTA, the PLA₂ inhibitor p-bromophenacyl bromide, and prolonged pretreatment with heat. Bovine serum albumin (1%) prevented the increase in metabotropic binding by PLA₂. Arachidonic acid failed to mimic the PLA₂ effect on metabotropic binding. These results indicate that PLA₂ can selectively modulate certain subtypes of excitatory amino acid receptors. This effect is due to the enzymatic activity but is probably not correlated with the formation of arachidonic acid metabolites. Independent of their possible physiological implications, our results provide the first autoradiographic evidence that an enzymatic treatment can selectively affect the binding properties of excitatory amino acid receptors in different regions of the CNS.     

Metamorphic Changes in Glycolipids and Myelin Proteins and 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Bullfrog and Axolotl Brains

Abstract: The metamorphic changes in levels of glycolipids and myelin proteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in the brains of bullfrog tadpoles, adult frogs, and axolotls were investigated, with particular emphasis on myelin maturation. The concentrations of cerebroside. sulfatide, and galactosyldiacylglycerol gradually increased from the onset of prometamorphosis throughout the active metamorphic period and then greatly increased after metamorphosis was completed. The ratio of glucocerebroside to galactocerebroside increased greatly in the prometamorphic period and then rapidly decreased to the frog level during the climax period. The fatty acid compositions of cerebroside and sulfatide showed a developmental change, with 24:1 being more predominant in the later metamorphic stage. The proportion of hydroxy fatty acids increased up to the onset of the prometamorphic stage and thereafter remained constant at ∼ 50% of the total. The CNP activity remained unchanged throughout metamorphosis at 60% that in frog myelin and increased in the adult frog. The composition of tadpole myelin proteins remained constant during metamorphosis, with large basic protein being the most abundant, and in the frog, proteolipid protein and large basic protein were present in comparable amounts. The two adult forms of axolotl, i.e., the neotenous and metamorphosed forms, exhibited almost identical myelin constituents, and CNP activity in the neotenous form amounted to one-fifth that in the bullfrog. These results indicate that active biosynthesis of myelin marker components occurs as metamorphosis proceeds, but more pronounced changes of myelin components occur after metamorphosis is completed.