Distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 in the gastrointestinal tract of the rat

Summary The distribution and characterization of the opioid octapeptide met⁵-enkephalin-arg⁶-gly⁷-leu⁸ (met⁵-enk-arg⁶-gly⁷-leu⁸) within the gastrointestinal tract of the rat has been determined by immunohistochemistry and radioimmunoassay by use of a newly developed antibody to met⁵-enk-arg⁶-gly⁷-leu⁸. With both techniques, met⁵-enk-arg⁶-gly⁷-leu⁸-immunoreactivity (met⁵-enk-arg⁶-gly⁷-leu⁸IR) was detected in all regions of the gastrointestinal (GI) tract except the esophagus. The highest concentration of immunoreactive met⁵-enk-arg⁶-gly⁷-leu⁸ was observed in the colon, while intermediate concentrations were found in the stomach, duodenum, jejunum, and ileum. Immunostained somata were observed chiefly in the myenteric plexus; immunostained processes were present primarily in the myenteric plexus and the circular muscle layer. This distribution pattern is similar to that previously observed with antiserum to met⁵-enkephalin-arg⁶-phe⁷ (met⁵-enk-arg⁶phe⁷). Chromatographic analysis of met⁵-enk-arg⁶-gly⁷leu⁸-immunoreactive peptides extracted from the GI tract revealed the presence of an immunoreactive peptide of high molecular weight which accounted for approximately three-quarters of met⁵-enk-arg⁶-gly⁷-leu⁸-IR in both stomach and colon. These findings suggest a role for peptides related to the octapeptide met⁵-enk-arg⁶-gly⁷-leu⁸ in the regulation of GI function.     

A Kinetic Analysis of 6-[18F]Fluoro-l-Dihydroxyphenylalanine Metabolism in the Rat

Abstract: A previous study of the metabolism of 6-[¹⁸F]-fluoro-l -3,4-dihydroxyphenylalanine (FDOPA) in rats pretreated with carbidopa contained information amenable to kinetic analysis. Using these data, tracer transfer coefficients and metabolic rate constants were estimated. After intravenous injection, FDOPA in circulation was O-methylated (k^D₀ = 0.055 min^?1), and the metabolite (O-methyl-FDOPA) escaped from plasma with a rate constant (k^M_?1) of 0.01 min^?1. The initial clearance of FDOPA to striatum (K^D₁) was 0.07 ml g^?1 min^?1, and the equilibrium distribution volume (V^D_e) was 0.67 ml g^?1. The initial clearance of O-methyl-FDOPA to striatum (K^M₁) was 0.08 ml g^?1 min^?1, and the equilibrium distribution volume (V^M_e) was 0.75 ml g^?1. The rate constant of FDOPA decarboxylation (k^D₃) was 0.17 min^?1 in striatum. The elimination of 6-[¹⁸F]fluorodopamine (FDA) from striatum suggested an apparent rate constant for monoamine oxidase activity (k′₇) of 0.055 min^?1. 6-[¹⁸F]Fluorohomovanillic acid (FHVA) was formed from 6-[¹⁸F]fluoro-l -3,4-dihydroxyphenylacetic acid with a rate constant (k₁₁) of 0.083 min^?1, and FHVA was eliminated from striatum (k₉) with a rate constant of 0.12 min^?1. The steady-state concentration ratios of FDA and its metabolites were shown to be functions of these rate constants.     

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Alterations in the Production of 14CO2 and [14C]Acetylcholine from [U-14C]Glucose in Brain Subregions Following Transient Forebrain Ischemia in the Rat

Abstract: The production of ¹⁴CO₂ and [¹⁴C]acetylcholine from [U-¹⁴C]glucose was determined in vitro using tissue prisms prepared from the dorsolateral striatum (a region developing extensive neuronal loss following ischemia) and the paramedian neocortex (an ischemia-resistant region) following 30 min of forebrain ischemia and recirculation up to 24 h. Measurements were determined under basal conditions (5 mMK⁺) and following K⁺ depolarization (31 mM K⁺). The production of ¹⁴CO₂ by the dorsolateral striatum was significantly reduced following 30 min of ischemia for measurements in either 5 or 31 mM K⁺ but recovered toward preischemic control values during the first hour of recirculation. Further recirculation resulted in ¹⁴CO₂ production again being reduced relative to control values but with larger differences (20–27% reductions) detectable under depolarized conditions at recirculation times up to 6 h. Samples from the paramedian neocortex showed no significant changes from control values at all time points examined. [¹⁴C]Acetylcholine synthesis, a marker of cholinergic terminals that is sensitive to changes in glucose metabolism in these structures, was again significantly reduced only in the dorsolateral striatum. However, even in this tissue, only small (nonstatistically significant) differences were seen during the first 6 h of recirculation, a finding suggesting that changes in glucose oxidation during this period were not uniform within all tissue components. The results of this study provide evidence that in a region susceptible to ischemic damage there were specific changes during early recirculation in the metabolic response to depolarization. This apparent inability to respond appropriately to an increased need for energy production could contribute to the further deterioration of cell function in vivo and ultimately to the death of some cells.     

Biochemical characterization of enkephalin-like immunoreactive peptides of adrenal glands

The total enkephalin-like immunoreactive peptide content of adrenal glands from dog, cattle, guinea pig and rat was investigated by radioimmunoassay using a (met⁵)-enkephalin antiserum. Dog adrenals contain the highest amount of peptides, cattle and guinea pig adrenals contain lesser amounts, and the rat adrenals had the least amount (0.05% that of the dog). Comparison of the (met⁵)-enkephalin content of the adrenal cortex and medulla with that of whole bovine adrenal gland indicates that the peptides are concentrated in the medulla. Analysis of the chromaffin granules from bovine adrenal medulla indicates this to be the primary storage site for (met⁵)-enkephalin-like peptides. Gel chromatography reveals a molecular heterogeneity of the immunoreactive peptides in all species tested; high molecular weight peptides account for a larger proportion of the immunoreactivity when compared with the low molecular weight peptides.     

Photophosphorylation in Scenedesmus in vivo: O2 evolution, ATP pools and transients, and phosphate binding during photoreduction of NO3−, NO2− and CO2

The relation between light-induced electron transport with NO₃^?, NO₂^? or CO₂ as acceptors, ATP pools and transients in dark-light-dark transitions, and phosphate uptake was examined in phosphorus-starved cells of Scenedesmus obtusiusculus Chod. Net O₂ evolution at saturating light was around 6 μmol × (mg chlorophyll × h)^?1 in the absence of any acceptor, but reached average rates of 21, 65 and 145 μmol × (mg chlorophyll × h)^?1 upon additions of 5 mM KNO₃, KNO₂ and KHCO₃, respectively. The apparent rate of photophosphorylation in transition experiments was only a few percent of the rate calculated from CO₂-dependent O₂ evolution. Blocking non-cyclic electron transport with DCMU inhibited phosphate assimilation, but acceleration of non-cyclic electron flow by addition of NO₃^? or NO₂^? did not stimulate phosphate assimilation as compared to the situation without an acceptor. A functional non-cyclic system might primarily be needed for an efficient shuttle transfer of ATP from the chloroplast to the cytoplasm. An inhibition of the non-cyclic system due to lack of reducible substrates accelerates the cyclic system and thus indicates a regulation mechanism between the two systems.     

Kinetics of NH4+ and K+ uptake by ectomycorrhizal fungi. Effect of NH4+ on K+ uptake

NH₄⁺ and K⁺ uptake experiments have been conducted with 3 ectomycorrhizal fungi, originating from Douglas fir (Pseudotsuga menziesii (Mirb.] Franco) stands. At concentrations up to 250 μM, uptake of both NH₄⁺ and K⁺ follow Michaelis-Menten kinetics. Laccaria bicolor (Maire) P. D. Orton, Lactarius rufus (Scop.) Fr. and Lactarius hepaticus Plowr. ap. Boud. exhibit K_m values for NH₄⁺ uptake of 6, 35, and 55 μM, respectively, and K_m values for K⁺ uptake of 24, 18, and 96 μM, respectively. Addition of 100 μM NH₄⁺ raises the K_m of K⁺ uptake by L. bicolor to 35 μM, while the V_max remains unchanged. It is argued that the increase of K_m is possibly caused by depolarization of the plasma membrane. It is not due to a competitive inhibition of K⁺ by NH₄⁺ since the apparent inhibitor constant is much higher than the K_m, for NH₄⁺ uptake. The possibility that NH₄⁺ and K⁺ are taken up by the same carrier can be excluded. The K_m, values for K⁺ uptake in the two other fungi are not significantly affected by 100 μM NH₄⁺. Except for a direct effect of NH₄⁺ on influx of K⁺ into the cells, there may also be an indirect effect after prolonged incubation of the cells in the presence of 100 μM NH₄⁺.     

Kinetic Studies of a (Na++ K++ Mg2+) ATPase in Sugar Beet Roots

Kinetic studies of a dithiothreitol treated membrane ATPase fraction from sugar beet roots led to the following conclusions: 1) In the presence of MgATP, Na⁺ and K⁺ stimulate the ATPase activity in different ways following simple Michaelis-Menten kinetics. Thus separate sites for Na⁺ and K⁺ are suggested. 2) In the absence of K⁺, Na⁺ acts as an uncompetitive modifier raising the apparent K_m and V_max for MgATP. 3) In the absence of Na⁺, K⁺ activates non-competitively with respect to MgATP. Thus K⁺ increases V_max but does not affect the apparent affinity constant. 4) K⁺ and Na⁺ double the rate constants. 5) In the presence of Na⁺ or K⁺, Mg²⁺ in excess acts as a weak inhibitor to Na⁺ and/or K⁺ activity. 6) The temperature-activity dependence in the 5–40°C interval shows biphasic Arrhenius plots with the transition point between 15–18°C. The activation energy is lowered at temperatures > 18°C.     

Opioid peptides are substrates for the bifunctional enzyme LTA4 hydrolase/aminopeptidase

We determined if any naturally occurring peptides could act as substrates or inhibitors of the bifunctional, Zn²⁺ metalloenzyme LTA₄ hydrolase/aminopeptidase (E.C. 3.3.2.6). Several opioid peptides including met⁵-enkephalin, leu⁵-enkephalin, dynorphin_1–6, dynorphin_1–7, and dynorphin_1–8 competitively inhibited the hydrolysis of L-proline-p-nitroanilide by leukotriene A₄ hydrolase/ aminopeptidase, consistent with an interaction at its active site. The enzyme catalyzed the N-terminal hydrolysis of tyrosine from met⁵-enkephalin with K_m =450 ± 58 μM and V_max =4.9 ± 0.6 nmol-hr⁻¹-ug⁻¹ and from leu⁵-enkephalin with K_m =387 ± 90 μM and V_max =6.2 ± 2.5 nmol-hr⁻¹-ug⁻¹. Bestatin, captopril and carnosine inhibited the hydrolysis of the enkephalins. It is noteworthy that the bifunctional catalytic traits of this enzyme include generation of an hyperalgesic substance, LTB₄, and inactivation of analgesic opioid peptides.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

Effects of W-7, W-5, Verapamil and Diltiazem on vacuolar proton transport. Comparison of vacuolar H+-ATPase and H+-PPase from roots of Zea mays

The vacuolar membrane of plant cells is characterized by two proton pumps: the vacuolar H⁺-ATPase (V-ATPase; EC 3.6.1.3) and the vacuolar H⁺-PPase (V-PPase; EC 3.6.1.1). Recently, Du Pont and Morrissey reported that Ca²⁺ stimulates hydrolytic activity of purified V-ATPase (Arch. Biochim. Biophys., 1992. 294: 341–346). Since this effect may be due to degradation during purification further investigation of Ca²⁺ regulation of native V-ATPase was done. However, native tonoplast membranes contain a Ca²⁺/H⁺ antiport activity, which interferes with effects of calcium ions on proton transport activity of vacuolar ATPase. Therefore, the effects of anti-calmodulin drugs (W-7, W-5, calmidazolium), and calcium channel antagonists (Verapamil, Diltiazem) on proton transport activities of the vacuolar-type H⁺-ATPase and H⁺-PPase in tonoplast enriched membrane vesicle preparations from roots of Zea mays L. were studied. The concentrations for half maximal inhibition of vacuolar H⁺-ATPase (H⁺-PPase) were: 71 (191) μM W-7, 470 (> 800) μM W-5, 26 (24) μM calmidazolium (= compound R 24571). 398 (700) μM Verapamil, and 500 (1 330) μM Diltiazem. Estimation of Hill coefficients (n_H) for the inhibition by Verapamil showed a further difference between the two vacuolar proton pumps (H⁺-ATPase, n_H= 2.02; H⁺-PPase, n_n= 0.96). The data indicate that the vacuolar H⁺-ATPase itself is affected by these chemicals. It is suggested that some biological activities of W-7, W-5, Verapamil, and Diltiazem are due to their effects on proton translocation by the vacuolar-type H⁺-ATPase.     

Metabotropic Glutamate Receptor in C6BU-1 Glioma Cell Has NMDA Receptor-Ion Channel Complex-Like Properties and Interacts with Serotonin2 Receptor-Stimulated Signal Transduction

Abstract: We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-d -aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Mg²⁺ and Ca²⁺. Pertussis toxin treatment abolished this glutamate-induced [Ca²⁺]_i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg²⁺, d -2-amino-5-phosphonovalerate (d -APV), HA-966, and MK-801, also inhibited the increase in [Ca²⁺]_i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg²⁺ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine₂ (5-HT₂) receptor signaling. EAAs inhibited 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (d -APV, MK-801, phencyclidine, and HA-966), but l -APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca²⁺. In addition, this inhibitory effect on 5-HT₂ receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT₂ receptor-mediated [Ca²⁺]_i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca²⁺ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C.     

Regional Distribution of Methionine-Enkephalin-Arg6-Phe7 in the Rat Brain: Comparative Study with the Distribution of Other Opioid Peptides

The distribution of the opioid peptide methionine-enkephalin-arginine6-phenylalanine7 (M-Enk-Arg6-Phe7) has been investigated in various structures of the rat brain by using a highly specific radioimmunoassay (RIA). Immunoreactive M-Enk-Arg6-Phe7 has been further characterized by high performance liquid chromatography. The levels of M-Enk-Arg6-Phe7 in various structures of the rat brain were compared with the levels of several other opioid peptides, including methionine-enkephalin (M-Enk), leucine-enkephalin (L-Enk), dynorphin 1-13, and alpha-neoendorphin, which were also measured by RIA. There was a close relationship between the distribution of M-Enk-Arg6-Phe7 immunoreactive material (ir), M-Enk ir, and L-Enk ir. The distribution of dynorphin 1-13 ir and alpha-neoendorphin ir appeared to be distinct from that of the enkephalin group. These results are in agreement with recent reports on the cloning and sequencing of the c-DNA coding for the prohormones, in which it has been hypothesized that M-Enk-Arg6-Phe7 and M-Enk are synthesized by the same precursor, called proenkephalin, and that dynorphin-related peptides and alpha-neoendorphin arise from a separate precursor, prodynorphin.     

Luteinizing hormone-releasing factor (LRF)-like immunoreactivity in rat pancreatic islet cells.

Luteinizing hormone-releasing factor (LRF)-like immunoreactive material was demonstrated by the three-layer immunoperoxidase method in formalin-fixed tissue sections of the rat pancreas. Anti-LRF antiserum was prepared in rabbits by immunizing with synthetic LRF coupled to bovine serum albumin (BSA). The immunoreactive site of LRF reacting with antiserum resided between residues Tyr⁵ and Gly¹⁰-NH₂. A positive staining reaction was observed in the islet cells with the use of anti-LRF antiserum after solid phase immunoadsorption with BSA, whereas no staining was observed when adjacent control sections were prepared with anti-LRF antiserum after immunoadsorption with an LRF-BSA conjugate, or with rabbit anti-oxytocin antiserum. LRF-like immunoreactive material was isolated from the rat pancreata by methanol extraction. This material coeluted with synthetic and hypothalamic LRF in cation exchange chromatography on carboxymethyl cellulose, and dilutions of it gave an inhibition curve parallel to that of synthetic LRF in radioimmunoassay. The concentration of LRF-like material in the rat pancreas is 1.1 pg/mg wet weight. These results suggest that LRF or a closely LRF-related peptide is shared by the central nervous system and the gastrointestinal tract.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C₄ (LTC₄) binding sites were identified in membrane preparations from human fetal lung. Specific binding of [³H]-LTC₄ represented 95 percent of total binding, reached steadystate within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC₄. Binding assays were performed at 4°C under conditions which prevented metabolism of [³H]-LTC₄ (80 mM serineborate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC₄ standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (K_d) of 26 + 6 nM and a density (B_max) of 84 ± 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [³H]-LTC₄ was displaced by LTC₄ and its structural analogs with inhibition constants (K_j) of 10 to 30 nM, whereas LTD₄, diastereoisomers of LTD₁, LTE₄ and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC₄. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [³H]-LTC₄ in human fetal lung membranes.     

Australian mulga ecosystems –13C and 15N natural abundances of biota components and their ecophysiological significance

Samples of recently produced shoot material collected in winter/spring from common plant species of mulga vegetation in eastern and Western Australia were assayed for ¹³C and ¹⁵N natural abundance. ¹³C analyses showed only three of the 88 test species to exhibit C₄ metabolism and only one of seven succulent species to be in CAM mode. Non-succulent winter ephemeral C₃ species showed significantly lower mean δ¹³C values (– 28·0‰) than corresponding C₃-type herbaceous perennials, woody shrubs or trees (– 26·9, – 25·7 and – 26·2‰, respectively), suggesting lower water stress and poorer water use efficiency in carbon acquisition by the former than latter groups of taxa. Corresponding values for δ¹⁵N of the above growth and life forms lay within the range 7·5–15·5‰. δ¹⁵N of soil NH₄⁺ (mean 19·6‰) at a soft mulga site in Western Australia was considerably higher than that of NO₃^– (4·3‰). Shoot dry matter of Acacia spp. exhibited mean δ¹⁵N values (9·10 ± 0·6‰) identical to those of 37 companion non-N₂-fixing woody shrubs and trees (9·06 ± 0·5‰). These data, with no evidence of nodulation, suggested little or no input of fixed N₂ by the legumes in question. However, two acacias and two papilionoid legumes from a dune of wind-blown, heavily leached sand bordering a lake in mulga in Western Australia recorded δ¹⁵N values in the range 2·0–3·0‰ versus 6·4–10·7‰ for associated non-N₂-fixing taxa. These differences in δ¹⁵N, and prolific nodulation of the legumes, indicated symbiotic inputs of fixed N in this unusual situation. δ¹⁵N signals of lichens, termites, ants and grasshoppers from mulga of Western Australia provided evidence of N₂ fixation in certain termite colonies and by a cyanobacteria-containing species of lichen. Data are discussed in relation to earlier evidence of nitrophily and water availability constraints on nitrate utilization by mulga vegetation.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.