Opioid Effects on [3H]Norepinephrine Release from Dissociated Embryonic Locus Coeruleus Cell Cultures

Abstract: The acute and chronic effects of opioid exposure on [³H]norepinephrine ([³H]NE) release were examined in cell cultures of embryonic rat locus coeruleus (LC). Initial morphological and biochemical characterization of the cultures indicated that the cells exhibited properties similar to those observed in situ. Specific [³H]NE uptake was saturable with a K _m value of 222 ± 52 n M . [³H]NE accumulated by LC cells was released in response to 20 m M K⁺ stimulation, in a calcium-dependent manner. Both components of neurotransmitter release, spontaneous and K⁺ evoked, were significantly inhibited by β-endorphin, with the latter being maintained in the presence of tetrodotoxin. The pharmacology of the opioid effect was consistent with that of μ-receptor activation. The effect of chronic exposure to the μ-selective agonist fentanyl (1 μ M ) was examined following 4 days of drug treatment. Although there was no significant effect of fentanyl on K⁺-evoked [³H]NE release, these cells were tolerant to the acute inhibitory effect of β-endorphin. These results indicate that this is an appropriate system for examining the effects of acute and chronic opioid treatment on noradrenergic cells in vitro. In addition, this system may be useful as a CNS model for examining mechanisms that underlie tolerance and dependence following chronic opioid exposure.     

Identification and Function of Glycine Receptors in Cultured Cerebellar Granule Cells

Abstract: Poly(A)⁺ mRNA was isolated from cultured mouse cerebellar granule cells and injected into Xenopus oocytes. This led to the expression of receptors that evoked large membrane currents in response to glycine. Current-responses were also obtained after application of β-alanine and taurine, but these were very low relative to that of glycine (maximal β-alanine and taurine responses were 8 and 3% of that of glycine, respectively). The role of glycine receptors on K⁺-evoked transmitter release in cultured cerebellar granule cells was also assayed. Release of preloaded d -[³H]aspartate evoked by 40 m M K⁺ was dose dependently inhibited by glycine, and the concentration producing half-maximal inhibition was 50 μ M. Taurine, β-alanine, and the specific GABA_A receptor agonist isoguvacine also inhibited K⁺-evoked release, and the maximal inhibition was similar for all agonists (˜40%). The EC₅₀ value was 200 μ M for taurine, 70 μ M for β-alanine, and 4 μ M for isoguvacine. Bicuculline (150 μ M ) antagonized the inhibitory effect of isoguvacine (150 μ M ) but not that of glycine (1 m M ). In contrast, strychnine (20 μ M ) antagonized the inhibitory effect of glycine (1 m M ) but not that of isoguvacine (150 μ M ). The pharmacology of the responses to β-alanine and taurine showed that these agonists activate both glycine and GABA_A receptors. The results indicate that cultured cerebellar granule cells translate the gene for the glycine receptor and that activation of glycine receptors produces neuronal inhibition.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.     

μ-Opioid Receptor Stimulation of Inositol (1,4,5)Trisphosphate Formation via a Pertussis Toxin-Sensitive G Protein

Abstract: The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both μ-and δ-opioid receptors), we demonstrated that fentanyl, a μ-preferring opioid, caused a dose-dependent (EC₅₀= 16 n M ) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1–2 min. This response was of similar magnitude (25.4 ± 0.8 pmol/mg of protein for 0.1 μ M fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 m M carbachol (18.3 ± 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole-(a δ antagonist), reversible. Further studies using [ d -Ala², MePhe⁴, Gly(ol)⁵]enkephalin and [ d -Pen^2,5]enkephalin confirmed that the response was specific for the μ receptor. Incubation with Ni²⁺ (2.5 m M ) or in Ca²⁺-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 μ M fentanyl, 26.9 ± 1.5 pmol/mg of protein; pertussis-treated plus 0.1 μ M fentanyl, 5.1 ± 1.3 pmol/mg of protein). In summary, we have demonstrated a μ-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca²⁺-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca²⁺, could play a part in the cellular mechanisms of opioid action.     

Evidence for κ- and μ-Opioid Receptor Expression in C6 Glioma Cells

Abstract: The astrocytoma cell line rat C6 glioma has been used as a model system to study the mechanism of various opioid actions. Nevertheless, the type of opioid receptor(s) involved has not been established. Here we demonstrate the presence of high-affinity U69,593, endomorphin-1, morphine, and β-endorphin binding in desipramine (DMI)-treated C6 cell membranes by performing homologous and heterologous binding assays with [³H]U69,593, [³H]morphine, or ¹²⁵I-β-endorphin. Naive C6 cell membranes displayed U69,593 but neither endomorphin-1, morphine, nor β-endorphin binding. Cross-linking of ¹²⁵I-β-endorphin to C6 membranes gave labeled bands characteristic of opioid receptors. Moreover, RT-PCR analysis of opioid receptor expression in control and DMI-treated C6 cells indicate that both κ- and μ-opioid receptors are expressed. There does not appear to be a significant difference in the level of μ nor κ receptor expression in naive versus C6 cells treated with DMI over a 20-h period. Collectively, the data indicate that κ- and μ-opioid receptors are present in C6 glioma cells.     

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (−)nicotine on the function of the NMDA-mediated modulation of [³H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [³H]DA release in rats chronically treated with vehicle (14 days) with an EC₅₀ of 13.1 ± 2.0 μM. The NMDA-evoked overflow of the [³H]DA in PFC nerve endings of rats treated with (−)nicotine was significantly lower (−43%) than in vehicle treated rats. The EC₅₀ was 9.0 ± 1.4 μM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [³H]DA overflow with an EC₅₀ of 14.5 ± 5.5 μM. This effect was significantly enhanced in chronically treated animals. The EC₅₀ was 10.5 ± 0.5 μM. The K⁺-evoked release of [³H]DA was not modified by the (−)nicotine administration. Both the changes of the NMDA-evoked [³H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (−)nicotine differentially affects the NMDA-mediated [³H]DA release in the PFC and NAc of the rat.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.     

Involvement of Protein Kinase C in γ-Aminobutyric Acid Release from Xenopus Oocytes Injected with Rat Brain mRNA

Abstract: Involvement of protein kinase C (PKC) in the release of γ-aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA-injected oocytes preloaded with [³H]GABA showed spontaneous release of [³H]GABA, ∼0.5% of GABA content per 1 min. Stimulation with either Ca²⁺ ionophore (A23187) or a high K⁺ concentration increased the release of [³H]GABA from slices of rat deep cerebellar nucleus and mRNA-injected oocytes but not from noninjected and water-injected oocytes. 12- O -Tetradecanoylphorbol 13-acetate (10–300 n M ) but not 4α-phorbol 12,13-didecanoate (300 n M ) potentiated the A23187-stimulated release of [³H]GABA from slices and from mRNA-injected oocytes, in a concentration-dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.     

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

Endogenous cannabinoid anandamide inhibits nicotinic acetylcholine receptor function in mouse thalamic synaptosomes

The effects of the endogenous cannabinoid anandamide [arachidonylethanolamide (AEA)] on the function of nicotinic acetylcholine receptor (nAChR) were investigated using the ⁸⁶Rb⁺ efflux assay in thalamic synaptosomes. AEA reversibly inhibited ⁸⁶Rb⁺ efflux induced by 300 μM ACh with an IC₅₀ value of 0.9 ± 2 μM. Pre-treatment with the cannabinoid (CB₁) receptor antagonist SR141716A (1 μM), the CB₂ receptor antagonist SR144528 (1 μM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA, suggesting that known CB receptors are not involved in AEA inhibition of nAChRs. AEA inhibition of ⁸⁶Rb⁺ efflux was not reversed by increasing acetylcholine (ACh) concentrations. In radioligand binding studies, the specific binding of [³H]-nicotine was not altered in the presence of AEA, indicating that AEA inhibits the function of nAChR in a non-competitive manner. Neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor, indomethacin, (5 μM) affected AEA inhibition of nAChRs, suggesting that the effect of AEA is not mediated by its metabolic products. Importantly, the extent of AEA inhibition of ⁸⁶Rb⁺ efflux was significantly attenuated by the absence of 1% fatty acid free bovine serum albumin pre-treatment, supporting previous findings that fatty acid-like compounds modulate the activity of nAChRs. Collectively, the results indicate that AEA inhibits the function of nAChRs in thalamic synaptosomes via a CB-independent mechanism and that the background activity of these receptors is affected by fatty acids and AEA.     

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF DMSP LYASE (DIMETHYLPROPIOTHETIN DETHIOMETHYLASE (4.4.1.3)) FROM THE RED ALGA POLYSIPHONIA PANICULATA1

Polysiphonia paniculata Montagne is an intertidal red alga known to produce large amounts of the compound dimethylsulfoniopropionate (DMSP). Conversion of this substrate into dimethylsulfide is accomplished in P, paniculata by an enzyme called DMSP lyase (dimethylpropiothetin dethiomethyla.se (4.4.1.3)). DMSP lyase has been purified and characterized from P. paniculata. Enzymie activity is found in two different proteins: the larger with a molecular weight of 9.26 ± 10⁴ daltons and the smaller with a molecular weight of 3.65 ± 10⁴ daltons. Specific activity of the enzyme is 526 μmols min⁻¹mg⁻¹ for the smaller protein a nd 263 μmols min ⁻¹ mg⁻¹ for the la rger protein. The Michaelis-Menten constant (K_m) is 72.8 μM ± 17.15 and the v_max is 1.62 μmols min⁻¹± 0.928 for the 92.6-kDa protein. The p1 of the larger protein is 5.8 and 5.9 for the smaller protein. Interaction with cysteine protease inhibitors L-trans-epoxysuccinyl-leucylamido (4-guanidino)-butane, dithiobis-(2-nitrobenzoate), or N -ethylmaleimide inactivated enzyme activity. The presence of either magnesium or calcium with DMSP lyase enhanced activity al concentrations between 20 and 40 μM but had little effect above these levels. Addition of the divalent chelators ethylenebis(oxyethylenenitrilo) tetraacetic acid and ethylenediaminetetraacetate decreased activity of the enzyme, but activity was restored when either chelator was removed and magnesium or calcium was added to the enzyme .     

Cyclic AMP Phosphodiesterase Activities in Xenopus laevis Oocytes

Cyclic AMP phosphodiesterase activity has been identified in full-grown Xenopus oocytes in vivo and in vitro. About 50% of the in vitro phosphodiesterase activity was present in the soluble fraction and 35% in a partially purified membrane fraction. Both activities exhibited high substrate affinity (Km about 10⁻⁶ M). Sucrose gradient fractionation revealed two forms of phosphodiesterase: a 5 S form (peak I) and a 6.5 S form (peak II). Treatment with trypsin led to the activation of the soluble enzyme with the transformation of peak II into peak I.
Ethylene glycol bis (β-aminoethyl ether)-N,N'-tetraacetic acid, calcium dependent regulator, and Fluphenazine did not influence the enzyme activities suggesting that the oocyte phosphodiesterases were not Ca²⁺-dependent. Intact oocytes were induced to mature by exposure to progesterone; their phosphodiesterase activities and distribution tested in vitro were comparable to those of untreated oocytes.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Neuronal Regulation of Interleukin 6 Secretion in Murine Spleen: Adrenergic and Opioidergic Control

Abstract: The PNS was anticipated to be involved in the modulation of immune responses. To study aspects of this neuronal-immune communication, a recently developed tissue slice method was used to study the effects of adrenergic and opioidergic transmitters on interleukin 6 (IL-6) secretion in the spleen. The α₂-adrenergic agonist p -aminoclonidine (10⁻⁷ M ) inhibited IL-6 secretion (control vs. p -aminoclonidine, 100.0 ± 4.76 vs. 59.3 ± 6.6% of control values; p < 0.001). The α₁-adrenergic agonist methoxamine (10⁻⁸ M ) also inhibited IL-6 secretion (100.0 ± 4.8 vs. 71.5 ± 3.8%; p < 0.001). The endogenous opioids β-endorphin (10⁻¹⁰ M ), methionine-enkephalin (10⁻⁹ M ), and leucine-enkephalin (10⁻⁹ M ) inhibited IL-6 secretion as well ( p = 0.0051, p = 0.0337, and p = 0.0226, respectively). Electrical stimulation of spleen slices inhibited IL-6 secretion (100.0 ± 4.3 vs. 56.7 ± 4.6% of control values; p < 0.001). The involvement of α-adrenergic and opioidergic molecules in this electrically induced inhibition was shown by the use of antagonists. Electrical inhibition of IL-6 secretion was attenuated by phentolamine (10⁻⁷ M ; p = 0.0345), by naloxone (10⁻⁶ M ; p = 0.0046), by cyprodime (10⁻⁸ M ; p = 0.0014), and by the combination of cyprodime (10⁻⁷ M ) plus phentolamine (10⁻⁸ M ; p < 0.0001). We conclude from the complementary studies that the inhibition of IL-6 secretion induced by electrical pulses was mostly mediated by α-adrenergic and μ-opioidergic endogenous transmitters.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

Accumulation of N-Arachidonoylethanolamine (Anandamide) into Cerebellar Granule Cells Occurs via Facilitated Diffusion

Abstract: N -Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [³H]AEA accumulation by cerebellar granule cells is dependent on incubation time ( t _1/2 of 2.6 ± 0.8 min at 37°C) and temperature. The accumulation of AEA is saturable and has an apparent K _m of 41 ± 15 µ M and a V _max of 0.61 ± 0.04 nmol/min/10⁶ cells. [³H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 µ M phloretin (57.4 ± 4% of control) in a noncompetitive manner. [³H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [³H]AEA accumulation is fairly selective for AEA among other naturally occurring N -acylethanolamines; only N -oleoylethanolamine significantly inhibited [³H]AEA accumulation at a concentration of 10 µ M . The ethanolamides of palmitic acid and linolenic acid were inactive at 10 µ M . N -Arachidonoylbenzylamine and N -arachidonoylpropylamine, but not arachidonic acid, 15-hydroxy-AEA, or 12-hydroxy-AEA, compete for AEA accumulation. When cells are preloaded with [³H]AEA, temperature-dependent efflux occurs with a half-life of 1.9 ± 1.0 min. Phloretin does not inhibit [³H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein-mediated transport process that has the characteristics of facilitated diffusion.     

Sodium Transport from Blood to Brain: Inhibition by Furosemide and Amiloride

Abstract: Brain sodium uptake in vivo was studied using a modified intracarotid bolus injection technique in which the uptake of ²²Na ⁺ was compared with that of the relatively impermeable molecule, [³H]l-glucose. At a Na ⁺ concentration of 1.4 m M , Na ⁺ uptake was 1.74 ± 0.07 times greater than l -glucose uptake. This decreased to 1.34 ± 0.04 at 140 m M Na ⁺, indicating saturable Na ⁺ uptake. Relative Na ⁺ extraction was not affected by pH but was inhibited by amiloride ( K _i= 3 ± 10⁻⁷ M ) and by 1 m M furosemide. The effects of these two inhibitors were additive. Brain uptake of ⁸⁶Rb ⁺, a K ⁺ analogue, was measured to study interaction of K ⁺ with Na ⁺ transport systems. Relative ⁸⁶Rb ⁺ extraction was also inhibited by amiloride; however, it was not inhibited by furosemide. The results suggest the presence of two distinct transport systems that allow Na ⁺ to cross the luminal membrane of the brain capillary endothelial cell. These transport systems could play an important role in the movement of Na ⁺ from blood to brain.     

Reproduction and fecundity of tucunaré, Cichla kelberi (Perciformes: Cichlidae), an exotic species in Três Marias Reservoir, Southeastern Brazil

To evaluate the reproductive cycle and fecundity of tucunaré ( Cichla kelberi Kullander & Ferreira, 2006), 697 specimens were captured in Três Marias Reservoir, São Francisco River, Brazil during 1994–1995 and 2005–2006. Reproductive activity was recorded throughout the sampling periods, with females exhibiting asynchronous oocyte development and multiple spawnings with a peak in September–October. Vitellogenic oocytes of the tucunaré were elliptical in shape with the longest diameter approximately 1230 μm and the shortest 700 μm, yolk globules with ellipsoid inclusions, lipid vesicles, small cortical alveoli and thin zona radiata (9.3 ± 2.0 μm thickness). Follicular cells were prisma-shaped (49.0 ± 16.4 μm) in the vegetative pole, progressively becoming cubic cells in the animal pole toward the micropyle. Histochemical analyses indicated the presence of mucosubstances in the outer zona radiata and follicular cells that could be contributors to egg adhesiveness. Batch fecundity ranged from 4450 to 13 900 oocytes for females 31.5–43.5 cm total length, respectively, and correlated to gonadal weight ( r ² = 0.80) and body weight ( r ² = 0.70). Mean relative fecundity was 10.6 vitellogenic oocytes per gram body weight. As tucunaré is an exotic piscivorous species well-adapted to the Três Marias Reservoir, the present work may be considered a contribution toward future strategies for population control.     

Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

Abstract: Structural elements of the rat μ-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn¹⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for μ/δ selectivity (Ile¹⁹⁸ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [³H]DAMGO (³H-labeled d -Ala², N -Me-Phe⁴,Gly-ol⁵-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn¹⁵⁰ in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β-endorphin_1–31, JOM-13, deltorphin II, dynorphin_1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁸ to Val resulted in a four- to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.