Opioid and Cannabinoid Receptors Share a Common Pool of GTP-Binding Proteins in Cotransfected Cells, But Not in Cells Which Endogenously Coexpress the Receptors

1. Opioid (, , ) and cannabinoid (CB1, CB2) receptors are coupled mainly toG_i/G_o GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins.2. The stimulation of [³⁵S]GTPS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of - and -opioid receptors led to stimulation of [³⁵S]GTPS binding by either -selective (DAMGO) or -selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes.3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either - or -opioid receptors.4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and -opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress - and -opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptorsubtype.5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.     

Regulation of Phospholipase C Activity by Calcium Ions and Guanine Nucleotide in the Normoxic Cat Carotid Body

The carotid bodies (CB) are a paired chemoreceptor organ located at the bifurcation of the common carotid arteries. High O₂ tension suppresses while low tension activates afferent carotid chemoreceptor activity and the chemoreflex ventilatory response in the cat. The intracellular mechanism of chemotransduction is till now unknown. Previously we have shown different activities of phospholipase C (PLC) in normoxic, hypoxic and hyperoxic cat carotid body. Now we have addressed the question whether calcium ions and G-protein could be regulators of the formation of lipid derived messenger molecules in the cat carotid body. To answer this question, the PLC acting against [³H] inositol-phosphatidylinositol (PtdIns) and [³H] inositol-phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P₂] in the cat CB were investigated using labelled phospholipids as a source of the substrate. CB homogenate was used as a source of the enzyme. The results indicate that PLC acting on PtdIns is Ca²⁺-dependent, in contrary to that acting on PtdIns(4,5)P₂ which remains active in the presence of 10 mM EGTA. PtdIns(4,5)P₂-PLC is stimulated by GTPS. In the presence of Ca²⁺, GTPS has a synergistic stimulatory effect. PLC acting on PtdIns is not activated by GTPS. In the presence of calcium ions dopamine and a nonhydrozylable analogue of acetylocholine, carbachol, have a small stimulatory effect of about 30 % on PLC acting on PtdIns(4,5)P₂. GTPS enhances this effect. These results allow us to suggest that there are two pathways of phosphoinositides degradation in the CB, one of them is regulated by calcium ions/PtdIns-PLC/, the other one by G-protein/PtdIns(4,5)P₂-PLC/.     

Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

The binding of [³⁵S]GTPS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 M. The specific binding was 98%. Different guanine nucleotides were all able to compete with [³⁵S]GTPS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [³⁵S]GTPS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [³⁵S]GTPS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPS, did not displace [³⁵S]GTPS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [³⁵S]GTPS binding was strongly Mg²⁺-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [³⁵S]GTPS in the presence of GTP may reflect functional G-protein activity.     

Preparation and purification of γγ enolase (neuron-specific enolase) using high performance anion exchange chromatography

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of enolase isoenzymes from human and beef brain extracts. In the first step, a crude enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of enolase by this method was 7–8 mg of pure enzyme per 100 g of brain.     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation

Myristoylated G_o was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G_o) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_o, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5"-3-O-(thio)triphosphate (GTPS) activation induced disaggregation of the G_o oligomers to monomers. The G_o was crosslinked by a cross-linker, N,N"-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G_o was pretreated with GTPS. Immunoblot analysis also demonstrated oligomerization of the purified G_o proteins and its disaggregation triggered by GTPS. These results provided direct evidence for the disaggregation–coupling theory and the disaggregation action of GTPS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.     

The Activities of Six Exo- and Endopeptidases in the Substantia Nigra,Neostriatum, and Cortex of the Rat Brain

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and -Glutamyl transpeptidase (GTP), and two endopeptidases: Postproline endopeptidase (PEP) and Trypsin-like peptidase (T-L P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) GTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a Trypsin-like peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.     

Dopamine Is a Natural Inverse Agonist in the Lymnaea Nervous System

Investigation of [³⁵S]GTPS binding with G proteins in plasma membranes of the molluscan nervous system demonstrated the existence of an inverse agonism phenomenon for dopamine.     

Effect of cytochrome P450 1A induction on oxidative damage in rat brain

Polycyclic and halogenated aromatic hydrocarbons (PAHs and HAHs) can enhance the generation of reactive oxygen species (ROS) by inducing cytochrome P450 1A (CYP 1A) in vivo and in vitro. While the brain is vulnerable to oxidative injury, whether or not CYP 1A induction in the brain can produce measurable levels of oxidative damage has not been reported. The objective of this study was to investigate the effect of this induction on oxidative damage to the CNS. Time course changes in rat brain CYP 1A activity were determined by measurement of ethoxyresorufin Odeethylase (EROD) activity in whole brain homogenates. Three days after exposure of rats to five daily injections of 3methylcholanthrene (3MC) an approximately sevenfold increase in EROD activity was observed. Hepatic levels were increased 60–100 fold. This increase in CYP 1A activity was not accompanied by increased protein or lipid oxidation as measured by tryptophan fluorescence and TBAR formation, or decreased glutamine synthetase (GS) activity. These findings indicate that if increased CYP 1A activity in the brain following 3MC treatment leads to increased ROS generation, the increase is insufficient to overwhelm the endogenous antioxidant defense system, produce detectable oxidative damage, and alter glutamate homeostasis.     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

Effect of Dietary Lipids on Phospholipase D Activity in Rat Brain

Phospholipase D (PLD) is emerging as a major player in many novel signaling pathways. Based on recent studies correlating membrane composition with enzyme function, we speculated that feeding of dietary lipids to the newborns has a major impact on brain PLD activity. To test this hypothesis, the rat dams were fed fat-free powder containing either safflower oil or fish oil, and a control powdered chow. The pups were weaned onto the diet and sacrificed at 30 days of age. PLD activity was measured by transphosphatidylation assays using rat brain membranes. This study shows that microsome GTPS-dependent PLD activity in rats fed safflower oil or fish oil was significantly reduced by 38% and 30% respectively compared to controls. Oleate-dependent PLD activity in the safflower oil group, however, was significantly increased by 38%. In contrast, synaptosome membrane (P2) GTPS-dependent PLD activity in rats consuming safflower oil was significantly increased by 29%, but there was no difference in oleate-dependent PLD activity. Likewise, no difference was observed in microsome oleate-dependent PLD and P2 GTPS-dependent PLD activity between the fish oil and the control groups. These results indicate that dietary lipid intake appears to modulate phospholipid metabolism and differential expression of PLD isozymes in the brain.     

Rat hepatocytes prepared without collagenase: Prolonged retention of differentiated characteristics in culture

Rat hepatocytes prepared by collagenase digestion or ED TA dissociation were examined in culture for comparison of culture stability and morphology, and retention of selected adult rat liver characteristics. Cells prepared by EDTA perfusion followed by Percoll cen trifugation were deemed to form confluent monolayer cultures more rapidly and monolayers remained intact for up to 21 days without signs of nonparenchymal cell growth or loss of primary hepatocyte appearance. The spectrally determined cytochrome P-450 content remained constant through eight days in culture. Collagenase prepared cells contained an identical amount of P-450 but within 72 hr lost greater than 80% of the spectrally detectable P-450. Glutathione (GSH) content was higher in the EDTA-prepared hepatocytes and remained constant with only a modest effect of transferrin and selenium (TI S) supplementation, while GSH levels in collagenaseprepared cells increased, thereafter decreased with time in culture and was dependent on TI S supplementation. Cells prepared with ED TA also displayed an increase in GSH efflux rate in response to chronic GSH depletion by ethacrynic acid. -Cystathionase (CNase) activity was retained at initial levels in ED TAprepared hepatocytes supplemented with TI S and declined only about 25% in unsupplemented cells. Collagenase-prepared cells lost 75% of CNase activity by 72 hr. The established marker of hepatocyte neoplastic transformation, -glutamyl trans-peptidase (GGT), increased rapidly in collagenase-prepared cells. The accumulation of GGT was slowed by T/S supplementation. GGT activity did not increase in EDTA-prepared hepatocytes. Evaluation of morphological and biochemical criteria suggest that hepatocytes prepared without collagenase present superior model systems for the study of biochemical events through more extended culture times.Abbreviations CNase -cystathionase - EDTA ethylenediamine tetraacetic acid - GGT -glutamyl transpeptidase - GSH reduced glutathione - GSSG oxidized glutathione - HEPES N-2-hydroxyethylpiperizine-N-2-ethane-sulfonic acid - SAH S-adenosylhomocysteine - SAM S-adenosylmethionine - T/S transferrin- and selenite-supplemented     

Subunit Composition of Nicotinic Acetylcholine Receptors in Neurons of the Rat Intracardiac Ganglia

The effect of the antibodies against 3, 4, 5, and 7 subunits of neuronal nicotinic acetylcholine receptors (nAChR) on ACh-induced shifts of the membrane potential (ACh potentials) in neurons of the intracardiac ganglia of the rat left atrium was studied using intracellular electrodes. It was found that (i) antibodies against each of the above subunits caused only a partial block of ACh potential; (ii) the blocking effect of each particular antibody could be observed only in some of the examined neurons; (iii) in all neurons under study the block caused by a single antibody was only partial; (iv) the blocking effects of two or more antibodies on the ACh potential recorded from the same neuron were additive. It was concluded that (1) each of the above nAChR subunits is present only in a part of the intracardiac neurons, and (2) each intracardiac neuron has nAChR composed of more than one subunit.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Polymerization of the Actin Component of the Cytoskeleton and Formation of the Geometry of a Cultured Nerve Cell (a Model Study)

Using mathematical modeling, we studied the biophysical aspects of the growth of the cell membrane and the growth of the actin network of the cytoskeleton of a neuron cultured on the rigid sublayer and the correlation between these processes. To describe the dynamics of the growth of the cytoskeleton limited by the cell membrane, we used the model of the thermal ratchet. Using the approaches of theoretical biophysics, we obtained a simple biophysical criterion that governs the selection of an alternative scenario of the formation of the cell, either growth of a single neurite or growth of a number of neurites. This criterion depends on the value of the adhesion between the cell and the substrate, the dimension of the actin monomer, and the thermal energy determining the frequency of thermal fluctuations of the cell membrane.     

Alterations in the expression of the β-cytoplasmic and the γ-smooth muscle actins in hypertrophied urinary bladder smooth muscle

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle -actin, shows over 200% increase in the -actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in -actin, since the 2D gel electrophoresis does not distinguish -smooth muscle actin from -cytoplasmic actin. The bladder smooth muscle -actin and the smooth muscle -actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the -cytoplasmic actin (37%) and a concomitant diminution in the -cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Influence of selenium on glutathione and some associated enzymes in rats with mammary tumor induced by 7,12-Dimethylbenz(a)anthracene

A recent finding in epidemiological and laboratory studies suggests that the ratio of selenium to glutathione is lower in breast cancer subjects than its control counterparts. Selenium, an antioxidant and anticarcinogen, can modify the status of glutathione and some associated enzymes by blocking peroxidation of lipids in membranes of cancer subjects. Studies were conducted using female albino rats of Wistar strain bearing mammary tumor induced by 7,12-dimethylbenz(a) anthracene to assess the biological role of selenium on some antioxidant enzymes associated with the maintenance of glutathione status. For induction of mammary tumor, 25 mg DMBA in a 1 ml emulsion of sunflower oil and physiological saline was injected subcutaneously to each rat. One group in each of control and tumor bearing rats, were fed 5 mg sodium selenite/kg diet from the day of tumor induction for 24 weeks. Increase in the reduced glutathione concentration was preceded by significant increase in the oxidized glutathione as well as in the activities of -glutamylcysteine synthetase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase by selenium administration in rats bearing tumor. However, selenium administration to rats bearing tumor decreased the activity of -glutamyl transpeptidase. These observations clearly demonstrate the influence of dietary selenium supplementation in correcting abnormal changes in glutathione turnover and some associated enzymes in tumor induced rats.