Chronic ethanol administration down-regulates cannabinoid receptors in mouse brain synaptic plasma membrane

The effects of chronic ethanol (EtOH) consumption on the central nervous system may be related in part to its action on biological membranes by altering various receptor functions. In the current study, we examined the effects of chronic EtOH (4 day inhalation) on cannabinoid receptors (CB1) labeled with [3H]CP55,940 in synaptic plasma membranes (SPM) isolated from mouse brain. Our results indicate the presence of a high level of CB1 receptors in controls (Bmax=12.0+/-0.3 pmol mg-1 protein) which decreased significantly (-58%) in SPM from mouse brain chronically exposed to EtOH. This effect occurs without any changes in the receptor affinity (Kd=2. 3+/-0.3 nM for control and 2.9+/-0.3 nM for EtOH group, P>0.05). Dissociation kinetic results showed a dissociation rate constant (K-1) of 0.09+/-0.01 min-1 for control and this dissociation rate constant decreased significantly in the chronic EtOH treated mice brain (0.05+/-0.01 min-1, P<0.05). The competition studies with anandamide resulted in a substantial decrease in [3H]CP55,940 binding in both the control and EtOH group, with a decrease (P<0.05) in the Ki values in the SPM of chronic EtOH exposed mice. Hill transformation analysis showed an nH close to one in control (0. 92+/-0.01). This did not change significantly after chronic EtOH (0. 95+/-0.01) administration, which indicates the existence of a single class of receptor for [3H]CP55,940 binding in SPM from control and EtOH treated mice. The observed down-regulation of CB1 receptors by chronic EtOH may indicate the involvement of cannabinoid receptors in EtOH tolerance and dependence.     

Lithium enhances muscarinic receptor-stimulated CDP-diacylglycerol formation in inositol-depleted SK-N-SH neuroblastoma cells

The psychotherapeutic action of Li+ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li+ on muscarinic receptor-stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo-inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 +/- 4 nmol/mg of protein. The addition of carbachol to [3H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [3H]CDP-DAG was completely blocked by the addition of 10 microM atropine, was not dependent on the presence of Li+, nor was it affected by co-incubation with myo-inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [3H]CDP-DAG was potentiated approximately 10-fold by Li+ and substantially reduced by coincubation with inositol. The formation of [3H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [3H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol > or = oxotremorine-M > bethanecol > or = arecoline > oxotremorine > pilocarpine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The deletion of six ORFs of unknown function from Saccharomyces cerevisiae chromosome VII reveals two essential genes: YGR195w and YGR198w

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2(3)H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol x h(-1) x (mg protein)(-1). A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Protein secretion in suspensions of isolated rat hepatocytes: no influence of acute ethanol administration

The effect of ethanol on the secretion of proteins was studied in hepatocytes isolated from 24-h fasted rats and from fed rats. Hepatocytes were isolated after collagenase disruption of the liver and incubated in a standard medium containing amino acids, bovine albumin, glucose, penicillin and streptomycin in HEPES buffer. Cell viability was determined by urea production and trypan blue exclusion. When studying protein export, a model had to be chosen in which the labeling is accomplished before the addition of the test agents. Cells were incubated with [3H]valine for 2.5 and 7.5 min followed by a 15-mM valine chase and the incubates were adjusted to final concentrations of ethanol of 50 mM, 100 mM, colchicine 5-50 microM or cycloheximide 18 microM. Cells and media were harvested at various times, and counts incorporated into medium and cell protein were determined. Cycloheximide inhibited protein synthesis by 99%, decreased protein secretion by 10-20%, but did not further inihibit protein labeling when given after the chase confirming the chase's effectiveness. Colchicine inhibited protein release by 27-54% depending on the dose. With control cells labeled protein and specifically albumin appeared in the medium 20 min from the start of the pulse and this release of protein was not inhibited by 50 mM or 100 mM ethanol incubated with cells from the same animal whether the donor has been fed or fasted. The values for the ethanol-treated cells ranged from 94.0 to 113% of the control values from 30 to 120 min after the addition of the pulse. Lactate levels were markedly elevated, and urea synthesis decreased in the presence of either 50 mM EtOH or 100 mM EtOH. Thus using a method that can distinguish the effect of ethanol on synthesis from secretion, it is concluded that acute exposure to EtOH does not interfere with protein secretion.     

Inhibition of nicotinic responses by cotinine in bovine adrenal chromaffin cells

We studied the effects of cotinine, the major metabolite of nicotine, on nicotine-induced increase in [3H]phorbol dibutyrate binding, activation of protein kinase C and [3H]noradrenaline release in primary cultured bovine adrenal chromaffin cells. Cotinine (1 mM, 15 min.) and nicotine (10 microM, 5 min.) increased the [3H]phorbol binding by 100% and 150%, respectively. Both a short-term (10 min.) and a long-term (24 hr) pretreatment with cotinine inhibited the effect of nicotine. A 24 hr pretreatment with cotinine (1 mM) also reduced the nicotine-induced increase in membrane-bound protein kinase C activity. Cotinine pretreatment (10 min.) dose-dependently inhibited the release of [3H]noradrenaline induced by nicotine and dimethylphenylpiperazinium. Cotinine pretreatment did not reduce the [3H]noradrenaline release induced by high extracellular potassium (56 mM) or veratrine (10 mg l-1). The results indicate that cotinine inhibits activation of protein kinase C and noradrenaline release induced by nicotinic agonists in primary cultures of bovine adrenal chromaffin cells. The results suggest that pre-existing cotinine could modify responses to acute exposure to nicotine in neural systems.     

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

Estimates of glycolysis, pyruvate (de)carboxylation, pentose phosphate pathway, and methyl succinate metabolism in incapacitated pancreatic islets

Pancreatic islets were cultured for 24 h in the presence of 1 mM glucose, which renders islets incapable of responding to glucose with insulin release. These islets were compared to islets maintained at 20 mM glucose for 24 h. Detritiation of [2-3H]glucose and [5-3H]glucose in 1 mM glucose islets was normal, suggesting that glucose transport and phosphorylation and all enzymes of glycolysis were not down-regulated in the incapacitated islets. 14CO2 formation from [U-14C]glucose and [6-14C]glucose was inhibited up to 80% and 14CO2 from methyl succinate was inhibited up to 60%, indicating that down-regulation at (a) mitochondrial site(s) might explain the incapacitated insulin release. 14CO2 formation from [3,4-14C]glucose (which becomes [1-14C]pyruvate) was decreased, indicating that the reaction catalyzed by pyruvate dehydrogenase was down-regulated. This decrease, however, was not as large as the decreases in 14CO2 formation from [U-14C]glucose, [2-14C]glucose (which becomes [2-14C]pyruvate), or [6-14C]glucose (which becomes [3-14C]pyruvate), indicating that other reactions were also down-regulated. 14CO2 formation from [1-14C]glucose was inhibited less than that from [6-14C]glucose in the incapacitated islets (34 vs 54%) and these rates indicated that flux of glucose through the pentose phosphate pathway was increased in the incapacitated islet, such that 29% (0.4 nmol of 1.4 glucose/100 islets/90 min) was metabolized via this pathway in the incapacitated islet but only 3.4% (0.1 of 2.9 nmol glucose/100 islets/90 min) was metabolized via the pentose pathway in the 20 mM glucose islets. With rates of 14CO2 evolved from glucose labeled at C2 and C6 and from methyl succinate labeled at C1 + C4 and C2 + C3 the 14CO2 ratio formula was used to calculate the ratios of carboxylated and decarboxylated pyruvate. Roughly equal amounts of pyruvate entered the citric acid cycle by each route in islets maintained for 24 h at 1, 5, or 20 mM glucose. The results indicate that decarboxylation and carboxylation of pyruvate were about equally suppressed in incapacitated islets and that direct inhibition of reactions of the cycle was unlikely. This is consistent with evidence which indicates that down-regulation of both pyruvate carboxylase and pyruvate dehydrogenase occurs in incapacitated islets, i.e., under long-term conditions that modify amounts of enzymes (MacDonald et al., 1991, J. Biol. Chem. 266, 22392-22397).(ABSTRACT TRUNCATED AT 400 WORDS)     

Congenital diaphragmatic hernia--a tale of two cities: the Toronto experience

1. Alveolar macrophages (AM phi) exhibit arginase activity and may, in addition, express an inducible form of nitric oxide (NO) synthase (iNOS). Both pathways may compete for the substrate. L-arginine. The present study tested whether two recently described potent inhibitors of liver arginase (N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AM phi and whether inhibition of arginase might affect L-arginine utilization by iNOS. 2. AM phi obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3 x 10(6) cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [3H]-L-arginine uptake (37 kBq, 0.1 microM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AM phi were incubated for 1 h with [3H]-L-arginine (37 kBq, 0.1 microM) and the accumulation of [3H]-L-citrulline (NOS activity) and [3H]-L-ornithine (arginase activity) was determined. 3. During 1 h incubation of rabbit AM phi with [3H]-L-arginine, no [3H]-L-citrulline, but significant amounts of [3H]-L-ornithine (150 d.p.m x 1000) were formed. N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine, present during incubation, concentration-dependently reduced [3H]-L-ornithine formation (IC50: 2 and 45 microM, respectively). 4. N omega-hydroxy-D,L-indospicine (up to 100 microM) had no effect on [3H]-L-arginine uptake into rabbit AM phi, whereas 4-hydroxyamidino-D,L-phenylalanine caused a concentration-dependent inhibition (IC50: 300 microM). 5. Rat AM phi, cultured in the absence of LPS, formed significant amounts of [3H]-L-citrulline and [3H]-L-ornithine (133 and 212 d.p.m x 1000, respectively) when incubated for 1 h with [3H]-L-arginine. When AM phi had been cultured in the presence of 0.1 or 1 microgram ml-1 LPS, the formation of [3H]-L-citrulline was enhanced by 37 +/- 8.3 and 99 +/- 12% and that of [3H]-L-ornithine reduced by 21 +/- 8.7 and 70 +/- 2.5%, respectively. 6. In rat AM phi, cultured in the absence or presence of LPS, N omega-hydroxy-D,L-indospicine (10 and 30 microM) greatly reduced formation of [3H]-L-ornithine (by 80-95%) and this was accompanied by increased formation of [3H]-L-citrulline. However, only 20-30% of the [3H]-L-arginine not metabolized to [3H]-L-ornithine after inhibition of arginase was metabolized to [3H]-L-citrulline, when the AM phi had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AM phi had been cultured in the presence of LPS (i.e. high level of iNOS), all the [3H]-L-arginine not metabolized by the inhibited arginase was metabolized to [3H]-L-citrulline. 7. In conclusion, N omega-hydroxy-D,L-indospicine is a potent and specific inhibitor of arginase in AM phi. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of L-arginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.     

Survey of antibodies against various infectious disease agents in racing camels in Abu Dhabi, United Arab Emirates

1. The roles of both Ca2+ and adenosine 3':5'-cyclic monophosphate (cyclic AMP) in carbachol and K(+)-stimulated [3H]-noradrenaline release from SH-SY5Y human neuroblastoma cells were examined. 2. Both carbachol and K+ caused a time- and dose-related stimulation of [3H]-noradrenaline release. The release event in perfused cells was monophasic. Half-maximum stimulation measured in statically incubated (3 min) cells was 38 +/- 4 microM and 63 +/- 4 mM respectively. K+ (100 mM, added)-evoked release was greater than that produced by carbachol (1 mM). 3. Both carbachol and K+ caused a time- and dose (measured at 3 min)-related stimulation of cyclic AMP formation with half-maximum stimulation occurring at 5 +/- 1 microM and 49 +/- 2 mM respectively. In contrast to its effects on release, carbachol produced a greater stimulation of cyclic AMP formation than K+. 4. K(+)-stimulated [3H]-noradrenaline release was entirely dependent on Ca2+ entry as 2.5 mM Ni2+ abolished release. However, carbachol-evoked (1 mM) release appeared to be unaffected by Ni2+ pretreatment. 5. These data suggest that in SH-SY5Y cells, elevated cyclic AMP levels are not directly involved in [3H]-noradrenaline release. In addition, carbachol-stimulated release is largely independent of extracellular Ca2+ possibly implying a role for intracellular stored Ca2+ in the release process.     

Lesbians, bisexual women, and body image: an investigation of gender roles and social group affiliation

In our previous study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane (SPM) (Basavarajappa et al., Brain Res. 793 (1998) 212-218). In the present study, we investigated the effect of chronic EtOH (4-day inhalation) on the CB1 agonist stimulated guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding in SPM from mouse. Our results indicate that the net CP55,940 stimulated [35S]GTP gamma S binding was increased with increasing concentrations of CP55,940 and GDP. This net CP55,940 (1.5 microM) stimulated [35S]GTP gamma S binding was reduced significantly (-25%) in SPM from chronic EtOH group (175 +/- 5.25%, control; 150 +/- 8.14%, EtOH; P < 0.05). This effect occurs without any significant changes on basal [35S]GTP gamma S binding (152.1 +/- 10.7 for control, 147.4 +/- 5.0 fmol/mg protein for chronic EtOH group, P > 0.05). Non-linear regression analysis of net CP55,940 stimulated [35S]GTP gamma S binding in SPM showed that the Bmax of cannabinoid stimulated binding was significantly reduced in chronic EtOH exposed mouse (Bmax = 7.58 +/- 0.22 for control; 6.42 +/- 0.20 pmol/mg protein for EtOH group; P < 0.05) without any significant changes in the G-protein affinity (Kd = 2.68 +/- 0.24 for control; 3.42 +/- 0.31 nM for EtOH group; P > 0.05). The pharmacological specificity of CP55,940 stimulated [35S]GTP gamma S binding in SPM was examined with CB1 receptor antagonist, SR141716A and these studies indicated that CP55,940 stimulated [35S]GTP gamma S binding was blocked by SR141716A with a decrease (P < 0.05) in the IC50 values in the SPM from chronic EtOH group. These results suggest that the observed down-regulation of CB1 receptors by chronic EtOH has a profound effect on desensitization of cannabinoid-activated signal transduction and possible involvement of CB1 receptors in EtOH tolerance and dependence.     

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.     

Acute effects of cell isolation on InsP profiles in adult rat cardiomyocytes

The isolation and culture of adult rat cardiomyocytes was shown to cause major changes in the contents of [3H]-labeled inositol phosphates and inositol phospholipids. Undigested heart tissue contained high levels of [3H]Ins(1,4,5)P3 (5364+/-800 ct/min/g tissue, 80+/-12 ct/min/mg protein) and mass content averaged 13.8 nmol/g tissue or 208+/-36 pmol/mg protein (mean+/-S.E.M., n=4). After collagenase digestion, [3H]Ins(1,4,5)P3 was undetectable and the mass content of Ins(1,4,5)P3 had decreased to 0.8+/-0.2 pmol/mg protein (mean+/-S.E.M., n=4, P<0.01). [3H]Ins(1,4)P2 was reduced by 80% and [3H]PtdIns(4,5)P2 by 90%. These profiles remained essentially unchanged when the isolated cells were maintained in culture for up to 24 h, even though the inositol phosphate response remained sensitive to norepinephrine. Similar to findings in intact tissue, the inositol phosphate response to norepinephrine in these cells was inhibited by neither U-73122 (5 microM) nor by neomycin (5 mM). By 48 h in culture, the relative levels of [3H]Ins(1,4,5)P3 and [3H]Ins(1,4)P2 had increased in relation to the total inositol phosphate content and responses appeared to better reflect intact tissue. However, while retaining insensitivity to neomycin, cells at 48 h were fully sensitive to U-73122 (5 microM). These data demonstrate that altered inositol phosphate responses are observed in adult cardiomyocytes from the time of isolation and that while the profiles change over time in culture, a pattern similar to that in intact heart is not re-established.     

Studies of persistent infection by Chlamydia trachomatis serovar K in TPA-differentiated U937 cells and the role of IFN-gamma

To investigate the effects of adenosine A1 receptor activation on energy metabolism and RNA and protein biosynthesis in central neurons, cultured neurons from the rat forebrain were exposed for 1 hr to 72 hr to various concentrations (10 nM-100 microM) of the selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) or the A1 receptor antagonist 8-cyclopentyltheophylline (CPT). At all concentrations tested, the adenosinergic compounds did not affect cell viability within 72 hr of treatment, except for CPT, which reduced viability by 19.7% when used at the concentration of 100 microM. Energy metabolism was analysed by studying the specific uptake of 2-D-[3H]deoxyglucose ([3H]2DG). Rates of RNA and protein biosynthesis were assessed by the measurement of [3H]uridine and [3H]leucine incorporation, respectively. Neuronal [3H]2DG uptake was increased by 16% (P < 0.01) after 8 hr in the presence of 100 microM CCPA, whereas 100 microM CPT for 24 hr also increased [3H]2DG uptake (8%, P < 0.01). At these concentrations, both ligands inhibited [3H]uridine incorporation after a 3-hr treatment by 92% and 30%, respectively. CCPA never altered [3H]leucine incorporation when compared to controls, and CPT significantly inhibited protein synthesis only at 10-100 microM. Additional experiments to analyse the influence of A1 ligands on the transport of [3H]2DG, [3H]leucine and [3H]uridine suggested that CCPA and CPT, which interact functionally with adenosine receptors by regulating cyclic AMP production in this model, are able to alter energy metabolism and RNA synthesis in central neurons in a nonspecific manner by interacting with glucose and uridine transporters.     

The Stimulated Raman Spectrum of Symmetric 13C Cyanogen, 13C2N2

BACKGROUND: The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). METHODS: CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. RESULTS: Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. CONCLUSIONS: Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.     

N-acetylaspartylglutamate acetoxymethyl triester (NAAG.AM) as a tool for loading the neuropeptides NAAG and succinimidyl-NAAG into intact cells: effect on [3H]-dopamine exocytosis

Although N-acetylaspartylglutamate (NAAG) is one of the neuropeptides found in highest concentrations in the mammalian central nervous system, its functional role in neuronal signaling has not been definitively established. In some neuronal populations, NAAG is concentrated in nerve terminals and thus, it may play a role in the cytoplasmic events underlying neurotransmitter exocytosis. In the present study we have validated the use of the synthetic derivative NAAG-acetoxymethyl triester (NAAG.AM) as a tool to increase the intracellular levels of the peptide and assessed the ability of NAAG to regulate [3H]-dopamine ([3H]-DA) secretion in PC12 cells. Enzymatic degradation of NAAG.AM by nonspecific brain esterases resulted in the progressive formation of NAAG and succinimidyl-NAAG (Asu-NAAG). However, only 8% of NAAG.AM was converted to NAAG. Significant amounts of NAAG (1 nmol/mg protein) were demonstrable in cultures of the neuroblastoma cell line N2A following incubation with NAAG.AM for 2 h, with the concentration of (Asu)-NAAG being at least 100-fold higher. The pheochromocytoma cell line PC12 was used to assess the influence of loaded NAAG derivatives on [3H]-DA exocytosis. Incubation with 0.1-1 mM NAAG.AM did not affect the basal efflux or total content of [3H]-DA. However, it induced a dose-dependent decrease of [3H]-DA secretion in response to 56 mM KCl depolarization reaching an inhibition of 49% with 1 mM NAAG.AM. In contrast, NAAG.AM did not affect secretion induced by the calcium ionophore A23187 (100 microM). The present study validates the use of NAAG.AM as a tool to load NAAG derivatives into intact cells and provides preliminary evidence for an intracellular role of the peptide.     

Effects of 9-aminocamptothecin on newly synthesized DNA in patient bone marrow samples

9-Aminocamptothecin (9-AC) inhibited cell growth and DNA synthesis in HCT 116 human colon cancer cells in a concentration- and time-dependent manner. Interference with nascent DNA chain elongation was monitored using pH step alkaline elution. After a 3-day 9-AC exposure, 38% (10 nM) and 53% (50 nM) of the total [3H]DNA eluted with pH steps 11.3-11.7, compared to 9% in control cells. Effects on nascent DNA integrity were also evaluated by fixed elution with pH 12.1 buffer. After a 3-day exposure to 9-AC, 27% (10 nM) and 82.5% (50 nM) of the total [3H]DNA eluted relative to control. Paired bone marrow samples were then obtained in 10 patients before treatment and between 42 and 72 h of a continuous i. v. infusion of 9-AC (35-74 microgram/m2/h for 72 h). The mononuclear cells were incubated with [3H]dThd for 2 or 4 h, and then analyzed using either pH step or fixed pH alkaline elution, respectively. In seven patients receiving >/=47 microgram/m2/h 9-AC, 4% +/- 1.5% (mean +/- SE) of the total [3H]DNA eluted with pH steps /=59 microgram/m2/h 9-AC (n = 7). Since hematological toxicity is dose limiting on this 9-AC schedule, these cellular pharmacodynamic studies provide evidence of a DNA-directed cytotoxic effect of 9-AC in a sensitive host target tissue.     

Enhanced cytotoxicity with interleukin-1 alpha and 5-fluorouracil in HCT116 colon cancer cells

Recombinant human interleukin-1 alpha (rIL-1 alpha), at concentrations that were not growth-inhibitory when given alone (100-10,000 U/ml), enhanced the growth inhibition resulting from a 72-h fluorouracil (FUra) exposure in HCT116 colon cancer cells. Median-effect analysis of clonogenic assays indicated that rIL-1 alpha, given 24 h prior to and following a 24-h exposure to FUra, increased lethality in a more than additive fashion. rIL-1 alpha did not appear to significantly affect [3H]-FUra metabolism, total [3H]-FUra-RNA incorporation or RNA retention after drug removal, inhibition of thymidylate synthase, or thymidine triphosphate pool depletion. During continuous exposure to rIL-1 alpha, transient stimulation of RNA and DNA synthesis was observed at 72 h, with a return to normal by 96 h. A 24-h exposure to 10 microM FUra altered the elution profile of newly synthesized DNA as monitored by pH step alkaline elution. An accumulation of lower-MW single-stranded DNA species was noted with FUra compared to control, accompanied by a significantly decreased proportion of DNA retained on the polycarbonate filter: 10% retained vs. 32% for control (P = 0.01). A 48-h exposure to rIL-1 alpha alone did not affect the elution profile of nascent DNA species, nor did it enhance the effects of FUra. Although FUra did not appreciably affect pulse [3H]-uridine incorporation into RNA for the initial 8-24 h of FUra exposure, progressive inhibition of net RNA synthesis was observed thereafter. FUra prevented the stimulatory effect of rIL-1 alpha on RNA synthesis, and net RNA synthesis was significantly inhibited (by 64-79% after 72 and 96 h) with the combination compared to rIL-1 alpha alone. Continuous exposure to 10 microM thymidine did not rescue cells from the lethality of FUra alone or the combination of FUra plus rIL-1 alpha, suggesting that depletion of deoxythymidine triphosphate as a consequence of thymidylate synthase inhibition was not the most important component of FUra toxicity. In contrast, 1 mM uridine provided partial protection against the toxicity of FUra alone or with rIL-1 alpha. Although uridine did not affect FUra metabolism, it decreased FUra-RNA incorporation by 42-60%, presumably as a consequence of the 2-fold expansion of UTP pools. [125I]-rIL-1 alpha binding was nonspecific; with a 24-h exposure, however, internalized [125I]-rIL-1 alpha exceeded cell surface-bound material by 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological and trophical effects of Saccharomyces boulardii on the small intestine in healthy human volunteers

Recently we investigated the mechanisms mediating the transport of valproic acid (VPA) between blood and brain. In one study efflux of valproic acid (VPA) from rabbit brain was inhibited by probenecid. Efflux of VPA decreased when probenecid was given intravenously but not when probenecid was given by ventriculocisternal (VC) perfusion indicating that the major site of probenecid-sensitive transport was at the brain capillary endothelium and not at the choroid plexus. In another study VPA transport into rat brain was inhibited by para-aminohippurate (PAH). The purpose of the present study were to determine (a) if the efflux of VPA from rabbit brain was also inhibited by PAH, and (b) whether efflux of VPA could occur at the choroid plexus via an PAH-selective transport system. Six control rabbits received VPA by intravenous infusion and tracer concentrations of [3H]VPA and [14C]PAH by VC perfusion. Rabbits in the PAH group (n = 6) received identical treatment with VPA, tracer concentrations of [3H]VPA and [14C]PAH and, in addition, received 20 mM PAH by VC perfusion. PAH had no effect on the VC extraction ratio of [3H]VPA or the steady-state brain concentration of intravenously administered VPA. It is concluded that the efflux of VPA at the rabbit blood-brain barrier is mediated by a transporter different from the PAH-like transporter responsible for the uptake of VPA into rat brain. In addition, the finding that VC perfusion with PAH had no effect on the VC extraction of [3H]VPA provides further evidence that the choroid plexus plays a negligible role in removal of VPA from the CNS.     

The high prevalence of bipolar II and associated cyclothymic and hyperthymic temperaments in HIV-patients

The role of K+ channels in mediating vasorelaxation induced by two prostacyclin analogues was investigated in guinea-pig aorta. Iloprost caused substantial relaxation of tissues contracted with phenylephrine or 25 mM K+ but not 60 mM K+. In endothelial-denuded tissues, maximal relaxations to iloprost, cicaprost or isoprenaline were inhibited by approximately 40-50% with tetraethylammonium or iberiotoxin, both blockers of large conductance Ca2+-activated K+ (BKCa) channels. In contrast, the response to forskolin, an activator of adenylate cyclase was marginally inhibited by tetraethylammonium. The K(ATP) channel blocker, glibenclamide significantly augmented the response to iloprost but not cicaprost. These effects were largely inhibited by the EP1 receptor antagonist, 8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid 2-[1-oxo-3(4-pyridinyl)propyl]hydrazide, monohydrochloride (SC-51089) and partially by indomethacin, suggesting that iloprost relaxation is counterbalanced by activation of EP1 receptors, in part through a constrictor prostaglandin. We conclude that BKCa channels play an important role in mediating the effects of iloprost and cicaprost and raises the possibility that cyclic AMP-independent pathways might be involved.     

On the high affinity binding site for [3H]-1,3-dipropyl-8-cyclopentylxanthine in frog brain membranes

1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax values were not significantly (P > 0.05) modified (0.261 +/- 0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4. The specific binding of [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively. 5. The concentration-inhibition curves of adenosine agonists and antagonists versus [3H]-DPCPX binding showed the following order of potencies: CPA> R-PIA~ NECA> S-PIA> > CGS 21680, for the agonists, and XAC ~-DPCPX> > XCC> PACPX, for the antagonists.6. The present results suggest that the adenosine binding site in the frog brain membranes is G-protein coupled, but that the antagonist affinities and the pharmacological profile is different from the Al or A2 adenosine receptors.