Opioidergic effects of nonopioid analgesics on the central nervous system

1. The analgesic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is partly due to the fact that they act upon the periaqueductal gray matter (PAG) and the rostral ventromedial medulla of the brain stem and thus activate the descending pain-control system, which inhibits nociceptive transmission at the spinal dorsal horn.2. The analgesic action of dipyrone (metamizol) and of lysine-acetylsalicylate (LASA), two well-known NSAIDs, whether microinjected into the PAG or given systemically, can be reverted by naloxone. Repeated administration of dipyrone or LASA induces tolerance to their antinociceptive effect, with cross-tolerance to morphine, and a withdrawal syndrome upon naloxone administration. Dipyrone tolerance can be reverted by proglumide, a cholecystokinin antagonist.3. These findings reveal a close association between the central action of NSAIDs and endogenous opioids.     

Evidence for a wide occurrence of proton-translocating pyrophosphatase genes in parasitic and free-living protozoa

The BAZF gene has recently been identified as a novel homologue of the BCL6 oncogene. Here we cloned the human BAZF gene using murine BAZF as a probe. The predicted amino acid sequence was 91% identical to that of murine BAZF. The BTB/POZ and zinc finger domains were almost completely conserved between human and murine BAZF. Fluorescence in situ hybridization analysis revealed that the human BAZF gene is located on chromosome 17p13.1. Although expression of human BAZF mRNA was ubiquitously detected in human tissues, abundant expression was detected in heart and placenta. BAZF mRNA was expressed in some immature B cell lines and erythroleukemia cell lines. The expression in a human erythroleukemia cell line, HEL cells, was upregulated during megakaryocytic differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate. These expression patterns of BAZF mRNA suggest that BAZF may regulate differentiation in stages or lineages that are different from those regulated by BCL6.     

Short-term stretch translocates the alpha-1-subunit of the Na pump to plasma membrane

Short-term (2–30 min) cyclic stretch activates the Na pump in cultured aortic smooth muscle cells (ASMCs). This effect of stretch involves the phosphotidylinositol 3-kinase (PI 3-kinase) participation. Presently, we investigated whether this stimulation is the result of translocation of Na⁺,K⁺-ATPase from endosomes to the plasma membrane. ASMCs were stretched 20% for 5 min using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na⁺,K⁺-ATPase α-1-subunit protein. Membrane marker enzyme, 5′ nucleotidase activity, and the early and recycling endosome markers Rab4 and Rab11 were used to verify the enrichment of these fractions. Stretch increased Na⁺,K⁺-ATPase α-1 expression in plasma membrane fractions and decreased it in endosomes. PI 3-kinase inhibitors LY294002 and wortmannin blocked the stretch-induced translocation of the Na⁺,K⁺-ATPase α-1-subunit. Rab4 and Rab11 were enriched in the endosomal fraction, whereas 5′ nucleotidase activity was enriched in the plasma membrane fraction. We conclude that stimulation of the Na pump activity by shortterm cyclic stretch is the result, at least in part, of transport of the α-subunit of the enzyme from endosomes to the plasma membrane.     

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Electrogenic Properties of the Sodium Pump in a Dynamic Model of Membrane Transport

The general purpose of this theoretical work is to contribute to understand the physiological role of the electrogenic properties of the sodium pump, by studying a dynamic model that integrates diverse processes of ionic and water transport across the plasma membrane. For this purpose, we employ a mathematical model that describes the rate of change of the intracellular concentrations of Na⁺, K⁺ and Cl⁻, of the cell volume, and of the plasma membrane potential (V _m ). We consider the case of a nonexcitable, nonpolarized cell expressing the sodium pump; Na⁺, K⁺, Cl⁻ and water channels, and cotransporters of KCl and NaCl in its plasma membrane. We particularly analyze here the conditions under which the physiological V _m can be generated in a predominantly electrogenic fashion, as a result of the activity of the sodium pump. A major conclusion of this study is that, for the cell model considered, a low potassium permeability is not a sufficient condition for a predominantly electrogenic generation of the V _m by the sodium pump. The presence of an electroneutral exchange of Na⁺ and K⁺ represents a necessary additional requirement. Received: 8 September 1999/Revised: 21 March 2000     

Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

Golgi alkalinization by the papillomavirus E5 oncoprotein

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H(+)-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pH(G)) was measured by ratio imaging. Whereas pH(G) of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH approximately 7.0). The Golgi buffering power and the rate of H(+) leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pH(G). We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.     

Ginsenoside Rc and Rg1 Differentially Modulate NMDA Receptor Subunit mRNA Levels after Intracerebroventricular Infusion in Rats

We investigated the influence of centrally administered ginsenoside on the regulation of mRNA levels of the family of NMDA receptor subtypes (NR1, NR2A, NR2B, NR2C) by in situ hybridization histochemistry in the rat brain. The ginsenosides Rc and Rg1, the major components of ginseng saponin, differentially modulate NMDA receptor subunit mRNA levels in rat brain following prolonged i.c.v.-infusion. Ginsenosides Rc or Rg1 (10 g/10 l/hr for 7 days) was infused through preimplanted cannulae connected to osmotic mini-pumps. The level of NR1 mRNA is significantly increased in temporal cortex, caudate putamen, hippocampus, and granule layer of cerebellum in Rg1-infused rats as compared to control group. The level of NR2A mRNA is elevated in the frontal cortex. In contrast, it was decreased in CA1 area of hippocampus in Rg1-infused rats. However, there was no significant change of NR1 and NR2A mRNA levels in Rc-infused rats. The level of NR2B mRNA is elevated in cortex, caudate putamen, and thalamus in both Rc- and Rg-infused rats. In contrast, NR2B level is decreased in CA3 in Rg1-infused rats. The level of NR2C mRNA is increased in the granule layer of cerebellum in only Rg1 but not Rc infused rats. These results show that structure difference of ginsenoside may diversely affect the modulation of expression of NMDA receptor subunit mRNA after infusion into cerebroventricle in rats.