Characterization of kappa opioid receptor mediated, dynorphin-stimulated [35S]GTPγS binding in mouse striatum for the evaluation of selective KOR ligands in an endogenous setting.

Differential modulation of kappa opioid receptor (KOR) signaling has been a proposed strategy for developing therapies for drug addiction and depression by either activating or blocking this receptor. Hence, there have been significant efforts to generate ligands with diverse pharmacological properties including partial agonists, antagonists, allosteric modulators as well as ligands that selectively activate some pathways while not engaging others (biased agonists). It is becoming increasingly evident that G protein coupled receptor signaling events are context dependent and that what may occur in cell based assays may not be fully indicative of signaling events that occur in the naturally occurring environment. As new ligands are developed, it is important to assess their signaling capacity in relevant endogenous systems in comparison to the performance of endogenous agonists. Since KOR is considered the cognate receptor for dynorphin peptides we have evaluated the selectivity profiles of dynorphin peptides in wild-type (WT), KOR knockout (KOR-KO), and mu opioid receptor knockout (MOR-KO) mice using [35S]GTPγS binding assay in striatal membrane preparations. We find that while the small molecule KOR agonist U69,593, is very selective for KOR, dynorphin peptides promiscuously stimulate G protein signaling in striatum. Furthermore, our studies demonstrate that norBNI and 5'GNTI are highly nonselective antagonists as they maintain full potency and efficacy against dynorphin signaling in the absence of KOR. Characterization of a new KOR antagonist, which may be more selective than NorBNI and 5'GNTI, is presented using this approach.

Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors: comparison with functional GTPγS assays.

BACKGROUND: Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [(35)S]GTPγS to G protein cannot be observed in many "non-functional" D3 expressing cell lines. NEW METHOD: The present work explores the use of an alternate method for assessing agonist activity, consisting of measuring the difference in agonist competition between [(3)H]spiperone bound to low-affinity states of the receptor and that with radioligand bound to high-affinity states (GTP shift assay). COMPARISON WITH EXISTING METHOD: The current study describes the determination of GTP shifts in [(3)H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ(35)S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope (35)S. CONCLUSION: The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [(35)S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-selective agonist screening. It may also be useful for other GPCRs circumventing the need for functional assays and offering the ability to detect agonist activity regardless of the particular signaling pathway.

Identification of a Nfs1p-bound persulfide intermediate in Fe-S cluster synthesis by intact mitochondria.

Cysteine desulfurases generate a covalent persulfide intermediate from cysteine, and this activated form of sulfur is essential for the synthesis of iron-sulfur (Fe-S) clusters. In yeast mitochondria, there is a complete machinery for Fe-S cluster synthesis, including a cysteine desulfurase, Nfs1p. Here we show that following supplementation of isolated mitochondria with [(35)S]cysteine, a radiolabeled persulfide could be detected on Nfs1p. The persulfide persisted under conditions that did not permit Fe-S cluster formation, such as nucleotide and/or iron depletion of mitochondria. By contrast, under permissive conditions, the radiolabeled Nfs1p persulfide was greatly reduced and radiolabeled aconitase was formed, indicating transfer of persulfide to downstream Fe-S cluster recipients. Nfs1p in mitochondria was found to be relatively more resistant to inactivation by N-ethylmaleimide (NEM) as compared with a prokaryotic cysteine desulfurase. Mitochondria treated with NEM (1 mM) formed the persulfide on Nfs1p but failed to generate Fe-S clusters on aconitase, likely due to inactivation of downstream recipient(s) of the Nfs1p persulfide. Thus the Nfs1p-bound persulfide as described here represents a precursor en route to Fe-S cluster synthesis in mitochondria.

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.

Distinct expression of cold receptors (TRPM8 and TRPA1) in the rat nodose-petrosal ganglion complex.

TRPM8 and TRPA1 are cold-activated transient receptor potential (TRP) cation channels. TRPM8 is activated by moderate cooling, while TRPA1 is activated by extreme, noxious cold temperatures. These cold receptors are expressed in different subpopulations of primary afferent neurons. TRPA1 is co-expressed in a subpopulation of somatosensory neurons expressing TRPV1, which is activated by heat. However, the distribution and co-expression of these channels in the nodose-petrosal ganglion complex, which contains the jugular (JG), petrosal (PG), and nodose ganglia (NG) (mainly involved in putative somatic, chemo- and somato-sensation, and somato and visceral sensation, respectively), remain unknown. Here, we conducted in situ hybridization analysis of the rat nodose-petrosal ganglion complex using specific riboprobes for TRPM8, TRPA1, and TRPV1 to compare the features of the cranial sensory ganglia. Hybridization signals for TRPA1 were diffusely observed throughout these ganglia, whereas TRPM8 transcripts were seen in the JG and PG but not in the NG. We retrogradely labeled cranial nerve X with Fast Blue (fluorescent dye) and found TRPM8 transcripts in the jugular-vagal ganglion but not the NG neurons. TRPA1 transcripts were not detected in TRPM8-expressing neurons but were present in the subpopulation of TRPV1-expressing visceral sensory neurons. Taken together, these findings support that in the vagal system the expression of cold-activated TRP channels differs between nodose- and jugular-ganglion neurons suggesting different mechanisms of cold-transduction and that the TRPA1 distribution is consistent with its proposed function as a cold-sensing receptor in the visceral system.

Radiolabelled proteomics to determine differential functioning of Accumulibacter during the anaerobic and aerobic phases of a bioreactor operating for enhanced biological phosphorus removal.

Proteins synthesized by the mixed microbial community of two sequencing batch reactors run for enhanced biological phosphorus removal (EBPR) during aerobic and anaerobic reactor phases were compared, using mass spectrometry-based proteomics and radiolabelling. Both sludges were dominated by polyphosphate-accumulating organisms belonging to Candidatis Accumulibacter and the majority of proteins identified matched closest to these bacteria. Enzymes from the Embden-Meyerhof-Parnas pathway were identified, suggesting this is the major glycolytic pathway for these Accumulibacter populations. Enhanced aerobic synthesis of glyoxylate cycle enzymes suggests this cycle is important during the aerobic phase of EBPR. In one sludge, several TCA cycle enzymes showed enhanced aerobic synthesis, suggesting this cycle is unimportant anaerobically. The second sludge showed enhanced synthesis of TCA cycle enzymes under anaerobic conditions, suggesting full or partial TCA cycle operation anaerobically. A phylogenetic analysis of Accumulibacter polyphosphate kinase genes from each sludge demonstrated different Accumulibacter populations dominated the two sludges. Thus, TCA cycle activity differences may be due to Accumulibacter strain differences. The major fatty acids present in Accumulibacter-dominated sludge include palmitic, hexadecenoic and cis-vaccenic acid and fatty acid content increased by approximately 20% during the anaerobic phase. We hypothesize that this is associated with increased anaerobic phospholipid membrane biosynthesis, to accommodate intracellular polyhydroxyalkanoate granules.

[35S]GTP gamma S binding studies of amphiphilic drugs-activated Gi proteins: a caveat.

This paper documents a serious problem met during the testing of Gi protein-activating properties of a new series of synthetic compounds by measuring the induced binding of [(35)S]GTPgammaS to different subtypes of Gi protein. The problem arose from the strong affinity between [(35)S]GTPgammaS and the tested compounds, that are characterized by several (2-4) positive charges and high lipophilicity. Apparently, such affinity yields insoluble, labelled complexes that, also in the absence of Gi protein, are retained on the filters and give rise to false positive results.

Biochemical characterization of rab proteins from Bombyx mori.

The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.

The spinal antinociceptive effects of endomorphins in rats: behavioral and G protein functional studies.

BACKGROUND: Endomorphin-1 and endomorphin-2 are endogenous peptides that are highly selective for mu-opioid receptors. However, studies of their functional efficacy and selectivity are controversial. In this study, we systematically compared the effects of intrathecal (i.t.) administration of endomorphin-1 and -2 on nociception assays and G protein activation with those of [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), a highly effective peptidic mu-opioid receptor agonist. METHODS: Male Sprague-Dawley rats were used. Acute and inflammatory pain models were used to compare the duration and magnitude of antinociception. Agonist-stimulated [(35)S]GTP gamma S binding was used to observe the functional activity at the level of the receptor-G protein in both spinal cord and thalamic membranes. In addition, antagonists selective for each receptor type were used to verify the functional selectivity of endomorphins in the rat spinal cord. RESULTS: After i.t. administration, endomorphin-1 and -2 produced less antinociceptive effects than DAMGO in the model of acute pain. Concentration-response curves for DAMGO-, endomorphin-1-, and endomorphin-2-stimulated [(35)S]GTP gamma S binding revealed that both endomorphin-1 and -2 produced less G protein activation (i.e., approximately 50%-60%) than DAMGO did in the membranes of spinal cord and thalamus. In addition, i.t. endomorphin-induced antinociception was blocked by mu-opioid receptor selective dose of naltrexone (P < 0.05), but not by delta- and kappa-opioid receptor antagonists, naltrindole and nor-binaltorphimine (P > 0.05). CONCLUSIONS: Endomorphins are partial agonists for G protein activation at spinal and thalamic mu-opioid receptors. Both in vivo and in vitro measurements together suggest that DAMGO is more effective than endomorphins. Spinal endomorphins' antinociceptive efficacy may range between 53% and 84% depending on the intensity and modality of the nociceptive stimulus.

Neuroanatomical mapping of juvenile rat brain regions with prominent basal signal in [(35)S]GTPgammaS autoradiography.

[(35)S]GTPgammaS autoradiography represents a powerful functional approach to detect receptor-dependent G(i/o) protein activity in anatomically defined brain structures. Inherent to this technique, however, is the notable basal signal evident in several brain regions in the absence of receptor stimulation by exogenously added agonist. In the rat brain, much of this basal labelling derives from tonic activation of adenosine A(1) and lysophosphatidic acid LPA(1) receptors in the gray and white matter regions, respectively. Despite the elimination of the two receptor activities, prominent basal [(35)S]GTPgammaS labelling is still evident in discrete brain structures, possibly reflecting regional enrichment of G(i/o) and/or constitutive receptor activity or the presence of still unknown endogenous ligands activating their orphan receptors. Here, the anatomical distribution of the enhanced basal signal was systematically mapped in brain sections of 4-week-old male Wistar rats. Regions with prominent basal [(35)S]GTPgammaS labelling represented neuroanatomically distinct structures, in particular various thalamic and hypothalamic nuclei. For instance, the paraventricular thalamic nucleus, the bed nucleus of the stria terminalis and the subfornical organ were highly labelled, as were the periaqueductal gray and the nucleus of the solitary tract. Pre-treatment with N-ethylmaleimide (NEM), an alkylating agent preventing all known receptor-driven G protein activity in cryostat sections markedly decreased the basal binding in all examined regions. In preliminary screening, selective antagonists for various brain-enriched G(i/o)-coupled receptors failed to suppress the basal signal in any of the studied regions.

Estrogen and progesterone modulate [35S]GTPgammaS binding to nociceptin receptors.

Sex steroids modulate reproduction by altering the response of steroid-activated opioid circuits in the hypothalamus and limbic system, by inducing release of endogenous opioids and activation of their cognate receptors. Many studies have concentrated on steroid regulation of exogenous opioid peptides, but steroids also have important actions on opioid receptors inducing receptor trafficking. Opioid receptors are G protein-coupled receptors and their activation catalyzes the exchange of GTP for GDP initiating intracellular signaling cascades. Kinetics of G protein activation were studied using [(35)S]GTPgammaS binding. Catalytic amplification, the number of G proteins activated per occupied receptor, was used as a measure of receptor/transducer amplification. The present study examined whether estrogen and progesterone treatment altered the kinetics of nociceptin opioid receptor (ORL1) in plasma membranes from the medial preoptic area and mediobasal hypothalamus. These hypothalamic regions are important in the gonadal steroid hormone regulation of sexual receptivity. In the mediobasal hypothalamus, estrogen increased ORL1 (B(max)) receptor number 2-fold and maximal GTPgammaS binding (E(max)) 3.9-fold. Subsequent progesterone treatment further increased ORL1 E(max )6.9-fold above baseline, despite a 2-fold decrease in the catalytic amplification factor. In the medial preoptic area, estrogen alone did not increase E(max), but both estrogen and progesterone were able to increase ORL1 B(max) 2.2-fold and E(max) 3-fold, despite having a 3-fold decrease in the catalytic amplification factor. These effects are interesting because they indicate actions of steroids that increase the number of ORL1 but decrease the catalytic amplification suggesting that the steroid effects on opioid receptors are complex and may involve modulation by other signals.

Binding of 5'-O-(2-thiodiphosphate) to rat brain membranes is prevented by diadenosine tetraphosphate and correlates with ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) activity.

The distribution of binding sites for [(35)S]5'-O-(2-thiodiphosphate) ([(35)S]ADPbetaS), a radioligand of P2Y(1,12,13) receptors, and of ecto-nucleotide pyrophosphatase phosphodiesterase activity were analyzed in the rat forebrain. Binding sites for the radilogand are widespreadly distributed in the rat forebrain, showing the highest density in hypothalamus. K(d) values were in the range 1-2 nM. Diadenosine tetraphosphate (Ap(4)A) and diethenoadenosine tetraphosphate, epsilon-(Ap(4)A), displaced the radioligand, indicating dinucleotide binding to ADPbetaS-recognizing P2Y receptors. Activity ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), able to hydrolyze Ap(4)A and other diadenosine polyphosphates, is also widely distributed through the rat forebrain, with the highest activity in hypothalamus. These results suggests that Ap(4)A signalling mediated by P2Y(1,12,13) receptors and enzymatically regulated by NPP1 activity may be particularly important in hypothalamus and add new support for neurotransmitter/neuromodulatory functions of diadenosine polyphosphates in brain.

Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I.

Complex I of the respiratory chain is composed of at least 45 subunits that assemble together at the mitochondrial inner membrane. Defects in human complex I result in energy generation disorders and are also implicated in Parkinson's disease and altered apoptotic signaling. The assembly of this complex is poorly understood and is complicated by its large size and its regulation by two genomes, with seven subunits encoded by mitochondrial DNA (mtDNA) and the remainder encoded by nuclear genes. Here we analyzed the assembly of a number of mtDNA- and nuclear-gene-encoded subunits into complex I. We found that mtDNA-encoded subunits first assemble into intermediate complexes and require significant chase times for their integration into the holoenzyme. In contrast, a set of newly imported nuclear-gene-encoded subunits integrate with preexisting complex I subunits to form intermediates and/or the fully assembly holoenzyme. One of the intermediate complexes represents a subassembly associated with the chaperone B17.2L. By using isolated patient mitochondria, we show that this subassembly is a productive intermediate in complex I assembly since import of the missing subunit restores complex I assembly. Our studies point to a mechanism of complex I biogenesis involving two complementary processes, (i) synthesis of mtDNA-encoded subunits to seed de novo assembly and (ii) exchange of preexisting subunits with newly imported ones to maintain complex I homeostasis. Subunit exchange may also act as an efficient mechanism to prevent the accumulation of oxidatively damaged subunits that would otherwise be detrimental to mitochondrial oxidative phosphorylation and have the potential to cause disease.

Pharmacokinetics and distribution of [35S]methylsulfonylmethane following oral administration to rats.

Methylsulfonylmethane (MSM) is a sulfur-containing compound found in a wide range of human foods including fruits, vegetables, grains, and beverages. More recently, it has been marketed as a dietary supplement worldwide. The objective of this study was to evaluate the pharmacokinetic profile and distribution of radiolabeled MSM in rats. Male Sprague-Dawley rats were administered a single oral dose of [35S]MSM (500 mg/kg), and blood levels of radioactivity were determined at different time points for up to 48 h. Tissue levels of radioactivity at 48 and 120 h and urine and fecal radioactivity levels were measured at different time points for up to 120 h following [35S]MSM administration to rats. Oral [35S]MSM was rapidly and efficiently absorbed with a mean tmax of 2.1 h, Cmax of 622 microg equiv/mL, and AUC0-inf of 15124 h.microg equiv/mL. The t1/2 was 12.2 h. Soft tissue distribution of radioactivity indicated a fairly homogeneous distribution throughout the body with relatively lower concentrations in skin and bone. Approximately 85.8% of the dose was recovered in the urine after 120 h, whereas only 3% was found in the feces. No quantifiable levels of radioactivity were found in any tissues after 120 h, indicating complete elimination of [35S]MSM. The results of this study suggest that [35S]MSM is rapidly absorbed, well distributed, and completely excreted from the body.

Antimalarial activity of allicin, a biologically active compound from garlic cloves.

The incidence of malaria is increasing, and there is an urgent need to identify new drug targets for both prophylaxis and chemotherapy. Potential new drug targets include Plasmodium proteases that play critical roles in the parasite life cycle. We have previously shown that the major surface protein of Plasmodium sporozoites, the circumsporozoite protein (CSP), is proteolytically processed by a parasite-derived cysteine protease, and this processing event is temporally associated with sporozoite invasion of host cells. E-64, a cysteine protease inhibitor, inhibits CSP processing and prevents invasion of host cells in vitro and in vivo. Here we tested allicin, a cysteine protease inhibitor found in garlic extracts, for its ability to inhibit malaria infection. At low concentrations, allicin was not toxic to either sporozoites or mammalian cells. At these concentrations, allicin inhibited CSP processing and prevented sporozoite invasion of host cells in vitro. In vivo, mice injected with allicin had decreased Plasmodium infections compared to controls. When sporozoites were treated with allicin before injection into mice, malaria infection was completely prevented. We also tested allicin on erythrocytic stages and found that a 4-day regimen of allicin administered either orally or intravenously significantly decreased parasitemias and increased the survival of infected mice by 10 days. Together, these experiments demonstrate that the same cysteine protease inhibitor can target two different life cycle stages in the vertebrate host.

5-HT-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes.

Receptor-mediated guanine nucleotide-binding regulatory protein (G protein) activation or functional coupling between receptors and G proteins has been investigated by means of agonist-induced [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding, especially for the receptor subtypes negatively coupled to adenylyl cyclase through Gi type G proteins. In the present study, 5-HT-stimulated [35S]GTPgammaS binding to rat stritatal membranes was pharmacologically characterized in detail with the help of an extensive series of 5-HT receptor ligands. The optimum experimental conditions for the concentrations of GDP, MgCl2 and NaCl in the assay buffer were initially determined, and the standard assay was performed with 20 microM GDP, 5 mM MgCl2 and 100 mM NaCl. The specific [35S]GTPgammaS binding was stimulated by several compounds that had been shown to be agonists at 5-HT(1B/1D) receptors. The negative logarithmic values of the concentration eliciting half-maximal effect (pEC50) for these agonists were significantly correlated with their pKi's reported in the previous study of 5-HT1B receptor binding in rat frontal cortical membranes. The increase in specific [35S]GTPgammaS binding in response to 1 microM 5-HT was potently inhibited by several 5-HT(1B/1D) receptor antagonists as well as beta-adrenoceptor antagonists such as S(-)-cyanopindolol. On the other hand, 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl (BRL15572), a selective antagonist against human 5-HT1D receptors, was inactive as an antagonist at least up to 1 microM. Additionally, the concentration-response curve for 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L694247) was shifted rightward in parallel by the addition of S(-)-cyanopindolol at concentrations of 10 and 100 nM, indicative of the competitive inhibitory manner. The specific [35S]GTPgammaS binding was reduced by 1'-methyl-5-([2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl)-2,3,6,7-tetrahydrospirospiro(furo[2,3-f]indole-3,4'-piperidine) (SB224289) and methiothepin in a concentration-dependent manner. The inhibitory curve by either compound was shifted to the right by 10 and 100 nM S(-)-cyanopindolol, suggesting that these two drugs behaved as inverse agonists at 5-HT1B receptors in the present functional assay system. 5-HT-stimulated [35S]GTPgammaS binding to rat striatal membranes serves as a simple but useful method of investigating the functional interaction between the native 5-HT1B receptors and their coupled G proteins in this brain region.

Contribution of the vagus nerve and lamina terminalis to brain activation induced by refeeding.

Following refeeding, c-fos expression is induced in a particular set of brain regions that include the nucleus of the solitary tract (NTS), parabrachial nucleus (PB), central amygdala (CeA), paraventricular hypothalamic nucleus (PVH), supraoptic nucleus (SON) and the circumventricular organs. Within the PVH, the expression is particularly intense in the magnocellular division of the nucleus and it is as yet not clear how this activation occurs. The respective contribution of the vagus afferents and lamina terminalis, which conveys signals entering the brain through the forebrain circumventricular organs, has been investigated in rats subjected to a unilateral cervical vagotomy (UCV) or a unilateral lesion of the fibres running within the lamina terminalis (ULT) and projecting to the neuroendocrine hypothalamus. UCV significantly decreased postprandial c-fos expression in the NTS, PB, CeA and parvocellular division of the PVH. In contrast, ULT impaired postprandial activation of the magnocellular neurons in the PVH and SON. The present study also characterized the types of neurons activated in the PVH and SON during refeeding. In the magnocellular regions, arginine-vasopressin (AVP) neurons were activated upon refeeding whereas there was no apparent induction of Fos expression in oxytocin cells. In the parvocellular PVH, postprandial Fos was induced only in 30% of the corticotrophin-releasing factor (CRF) and AVP neurons. The results of the present study suggest that the postprandial activation of the brain requires the integrity of both the vagal- and lamina terminalis-associated pathways.

Reduced kappa-opioid activity in a rat model of cholestasis.

Increased endogenous opioid activity has been implicated in cholestatic pruritus. In the present study, we have further defined the involvement of opioids in cholestasis. Rats underwent either bile duct ligation or a sham operation. Five days after surgery, brains were removed and agonist-stimulated [35S]GTPgammaS binding was measured in ten brain regions. Serum endomorphin-2, leu-enkephalin and dynorphin A levels were measured using ELISA on day five. Microdialysis to the dorsal hypothalamic area was conducted in the same animal before and after cholestasis. Dialysate endomorphin-1, leu-enkephalin and dynorphin A levels also were measured. Delta- and kappa-stimulated binding was significantly decreased in cholestasic animals compared to controls in the dorsal hypothalamic area. The serum dynorphin A level was lower in the cholestasic group than in controls (2.56+/-0.09 and 3.29+/-0.22 ng/ml, respectively, P<0.01). We propose that pruritus in cholestasis may result from an impaired balance between mu- and kappa-opioid systems.

G protein-coupled galanin receptor distribution in the rat central nervous system.

The action of galanin in the central nervous system is mediated by at least three galanin receptor subtypes (GalR1, GalR2 and GalR3) which belong to the family of G protein-coupled receptors. GalR1 and GalR2 are coupled to G(i/o) proteins, although the latter may also be coupled to G(q/11) proteins. The aim of the present study was to identify the anatomical distribution and quantify the density of GalRs coupled to G proteins. The galanin (10(-6) M) stimulated guanosine 5'-(gamma-[35S] thio)triphosphate binding assay was used in tissue sections from the rat brain. Maximal percentages of stimulation over basal levels were found in the anterior olfactory nucleus and in the lateral olfactory tract nucleus ( approximately 54%). High levels of stimulation were recorded in diverse hypothalamic nuclei (16-28%), in the amygdala (central amygdaloid nucleus, 40%), in the spinal trigeminal tract (23%) and in layers 1-2 of the spinal cord (26%). Moderate binding stimulation (5-13%) was observed in thalamus, substantia nigra pars compacta, parabrachial nucleus, locus coeruleus and dorsal raphe nucleus. The lowest stimulation induced by galanin was recorded in diverse areas of the cortex, striatum, hippocampus and substantia nigra pars reticulata. The results show an anatomical distribution similar to that described for GalR1. However, in diverse brain areas, in which a high density of these receptors has previously been reported, only a moderate coupling to G proteins was found. These findings would suggest that the efficacy of galanin to induce an effective coupling of its receptors to G proteins could be different depending on the brain area.

Modelling the fate of sulphur-35 in crops. 1. Calibration data.

Gas-cooled nuclear power plants in the UK release sulphur-35 during their routine operation. The gas is in the form of COS which can be readily assimilated by vegetation. It is therefore necessary to be able to model the uptake of such releases in order to quantify any potential contamination of the food chain. To develop such models experimental data are required. A series of experiments was undertaken to determine the rate of deposition, the partition and subsequent loss of sulphur-35 in crops exposed to CO(35)S. The mass normalised deposition rate was similar for the range of crops tested, while the partition of the (35)S paralleled the growth of crop components. There was no significant loss of radioactivity other than that expected from radioactive decay.