Cannabinoid receptors CB1 and CB2 form functional heteromers in brain

Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.     

Kappa-opioid receptor in the rodent hippocampus: a comparative immunocytochemical study in the rat,guinea pig,hamster and gerbil

Pre-embedding light microscopic immunocytochemistry, using a monoclonal antibody (mAb-KA8) raised against a frog brain kappa receptor preparation, recognising selectively the kappa-opioid receptor, was used for studying the occurrence, distribution, and species-specificity of the kappa-opioid receptor in the hippocampal formation of four rodent species (rat, guinea pig, hamster and gerbil). MAb-KA8 immunoreactivity was detectable in the rat, hamster and gerbil hippocampus, however the distribution of the labelled structures was heterogeneous. In the rat and hamster the hilus of dentate gyrus and the stratum oriens of the CA1 area contained immunoreactive cell bodies and proximal dendrites. In the gerbil mAb-KA8 immunopositive cell bodies were recognisable in the stratum radiatum of the CA1 and CA3 areas and in the subiculum. In the hamster varicose axon-like elements were also detected in the CA3 pyramidal layer. With the mAb-KA8 antibody there was no detectable kappa opioid receptor labelling in the hippocampus of the guinea pig. The results confirm the high degree of species-specific heterogeneity characterising the distribution of opioid peptides and their receptors in the hippocampal formation. The receptor was found in most cases postsynaptically, however in the hamster the immunopositive axons may refer to a presynaptic localisation.     

Calbindin D-28k Immunoreactivity and Its Protein Level in Hippocampal Subregions During Normal Aging in Gerbils

The hippocampus is associated with learning and memory function and shows neurochemical changes in aging processes. Calbindin D-28k (CB) binds calcium ion with a fast association rate. We examined age-related changes in CB immunoreactivity and its protein level in the gerbil hippocampus during normal aging. In the hippocampal CA1 region (CA1) and CA2, CB immunoreaction was found in some neurons in the stratum pyramidale (SP) at postnatal month 1 (PM 1). CB immunoreactivity in neurons was markedly increased at PM 3. Thereafter, CB immunoreactivity was decreased with time: CB-immunoreactive (⁺) neurons were fewest at PM 24. In the CA3, a few CB⁺ neurons were found only in the SP at PM 1 and in the stratum radiatum at PM 18 and 24. In addition, mossy fibers were stained with CB at PM 1. CB immunoreactivity in mossy fibers was markedly increased at PM 3, thereafter it was decreased with time. In the dentate gyrus, many granule cells (GC) in the granule cell layer were stained with CB at PM 1. CB immunoreactivity in GC was markedly increased at PM 3, thereafter CB immunoreactivity was decreased with time. In Western blot analysis, CB protein level in the gerbil hippocampus was highest at PM 3, thereafter CB protein levels were decreased with time. This result indicates that CB in the gerbil hippocampus is abundant at PM 3 and is decreased with age.     

Immunohistochemical demonstration of serotonin-containing nerve fibers in the mammalian hippocampal formation

The distributional pattern of serotonin-immunoreactive nerve fibers in the hippocampal formation of six different mammalian species (rat, chipmunk, hamster, cat, dog and monkey) was studied in detail by means of a modified peroxidase-antiperoxidase immunohistochemical method, using a specific serotonin antiserum. Furthermore, the density of varicosities distributed in each layer of the hippocampus was semiquantitatively analyzed. In all species investigated, the routes of serotonin fibers entering the hippocampal formation were found to be almost the same. These fibers were extensively distributed throughout the hippocampal formation, and had a characteristic arrangement corresponding to the laminar structure of this region. A dense innervation by varicose serotonin fibers was found in the stratum lacunosum-moleculare, but a few serotonin fibers were also distributed in the stratum lucidum of the CA2 and CA3 fields. The stratum pyramidale and the granule cell layer of the dentate gyrus contained a small number of serotonin fibers. The concentration and the direction of serotonin fibers were different in each area of each animal. Two peculiar observations should be stressed: (1) in the rat, the strata oriens and radiatum of CA2 and CA3 as well as the stratum lacunosum-moleculare displayed abundant serotonin fibers; (2) in the dog, abundant serotonin fibers were diffusely distributed in the CA1 field except for the stratum pyramidale and the most dense concentration of serotonin fibers was seen in the stratum oriens of CA3. The present study provides a morphological basis for further study of the functional significance of serotonin in the limbic system.     

Association of fibronectin-like antigens in chromatin preparations from rat hepatoma cells

High molecular weight hepatoma-associated nonhistone chromosomal proteins (NHPs) in transplantable rat hepatoma cells were reported previously from this laboratory. A cDNA library prepared from Morris hepatoma 7777 cells was screened with the polyclonal antibodies against hepatoma NHPs and a positive cDNA clone (lambda P2A1) was isolated. DNA sequence analysis revealed that the cDNA clone was identical to that of rat fibronectin (FN). The polyclonal antibodies against hepatoma NHPs were shown to bind specifically to both rat plasma FN and the fusion proteins encoded by lambda P2A1. A monoclonal antibody specific to rat plasma FN also recognized high molecular weight antigens of hepatoma NHPs in a pattern similar to that demonstrated with the polyclonal antibodies. These results suggest the existence of FN or FN-like antigens in the chromatin preparations from rat hepatoma cells. The antigenic proteins are localized in the nuclei of neoplastic foci of liver undergoing hepatocarcinogenesis.     

Characterization of the estrogen-induced lipoprotein receptor of rat liver

The ethinyl estradiol-induced lipoprotein receptor of rat liver was purified and characterized. Liver membranes were prepared from ethinyl estradiol-treated rats, solubilized, and subjected to DEAE chromatography. A fraction with a high specific activity for low density lipoprotein (LDL) binding was isolated and used to immunize mice. Hybridomas were prepared from their spleen cells, and a clone that secreted an IgG antibody, which cross-reacted with an ethinyl estradiol-induced protein of the same molecular weight as the bovine adrenal LDL receptor, was expanded. This antibody, designated P1B3, immunoprecipitated the induced lipoprotein receptor. P1B3 was used to purify the receptor, and a polyclonal antibody was raised against the pure protein. This antibody recognized a protein of similar molecular weight in rat liver, adult dog liver, and human skin fibroblasts, thus demonstrating that the induced rat lipoprotein receptor was related to the LDL receptor of other species. This receptor is present in normal rat liver, and its content is reduced by feeding an atherogenic diet, but not by feeding a diet containing 0.5% cholesterol. Moreover, cholestyramine supplementation of the diet did not induce the receptor on liver membranes. The polyclonal antibody could prevent the binding of LDL to liver membranes from control or ethinyl estradiol-treated rats. It decreased chylomicron remnant binding to membranes from ethinyl estradiol-treated membranes, but did not affect chylomicron remnant binding to liver membranes of untreated rats, a result compatible with the existence of a distinct receptor for these latter particles. The amount of LDL receptor-independent, specific remnant binding was the same in both control and ethinyl estradiol-treated rats. This is consistent with the concept that the remnant receptor is not regulated by this treatment. Based on the above, we conclude that the ethinyl estradiol-induced lipoprotein receptor of rat liver is biochemically and immunologically similar to the LDL receptor of other species. It is present on the liver of normal adult rats and could account for LDL as well as beta VLDL and HDLc removal. Although it may contribute to chylomicron remnant removal, there appears to be a second unrelated receptor or process which recognizes this lipoprotein.     

NPR1多肽抗体的制备和应用

根据NCBI GenBank中报道的NPR1一级结构信息,采用Blastn、Blastx、ExPASy和Protean等软件进行序列同源性和抗原性指数分析,获得三段序列特异性较高的多肽,并从中优选一段序列特异性多肽,采用9-氟甲氧羰基固相合成法获得序列特异性最好的多肽,采用HPLC和LC-MS测定合成多肽的浓度和分子量,试验表明目的多肽纯度达88%、目的多肽分子量为1.92234 kD。采用碳化二亚胺法将多肽与KLH进行偶联获得免疫原Pep-KLH,并将其免疫新西兰大白兔以获得抗血清和多克隆抗体,采用ELISA和Western blotting测定其效价和特异性,经ELISA检测表明抗血清和多克隆抗体可与Pep发生特异性免疫反应,经Western blotting试验表明抗血清和多克隆抗体可识别烟草叶片特异性条带,其相对分子量为65 kD,与预测分子量相符,表明利用该方法制备的NPR1多肽抗体具有较高特异性和灵敏度。     

Comparative mapping of opioid receptors and enkephalin immunoreactive nerve terminals in the rat hippocampus. A radiohistochemical and immunocytochemical study

Opioid receptors can be localized to the hippocampal formation of the rat by autoradiography. The binding of 3H-enkephalinamide to fixed and mounted tissue sections has all the characteristics associated with binding to opioid receptors. It is saturable, of high affinity and displays stereospecificity. The opioid receptor distribution shows striking regional variation throughout the hippocampal formation. Areas with high density include the pyramidal cell layer of both regio superior (CA1) and regio inferior (CA3), stratum moleculare of the hippocampus, the cell layer of subiculum, the superficial part of presubiculum and the deep layer (VI) of the medial and lateral entorhinal cortices. Areas with low to medium densities include regions corresponding to the dendritic field of the pyramidal cells (str. oriens, str. radiatum and the mossy fiber zone), the dentate granule cell layer and the molecular layer of the dentate area. Enkephalin-like immunoreactivity is detected in both intrinsic neuronal systems: 1) the mossy fibers which terminate on the proximal part of the CA3 pyramidal cell dendrites and on CA4 pyramidal cells, 2) cell bodies with multiple short processes, probably interneurons, dispersed throughout the hilus of the dentate area, the pyramidal cell layer of hippocampus, the str. radiatum, and occasionally in the str. moleculare and in the str. oriens, and extrinsic neuronal systems: 1) the lateral perforant path and 2) the lateral temporo-ammonic tract. Thus, the hippocampus contains intrinsic systems of enkephalin-like immunoreactive nerve terminals which may exert their effect on the opioid receptors with a localization corresponding to the pyramidal cells and their apical dendrites. Extrinsic enkephalinergic systems corresponding to the terminal fields of the lateral perforant path and the temporoammonic tract, both of entorhinal origin, may influence the opioid receptors located in the molecular layer of the dentate area, and in the molecular layer of the hippocampus and the subiculum. Thus, the enkephalin-like immunoreactive nerve terminals are all located in areas which contain opioid binding sites. This suggests that the "opioid peptide-opioid receptor" systems may regulate hippocampal neuronal activity via neurotransmission or neuromodulation. However, a high or medium number of opioid binding sites occur over the pyramidal cell bodies and the dentate granule cell bodies, and these opioid binding sites are not in close contact with the major enkephalinergic systems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

Cloning and molecular characterization of the rat CB2 cannabinoid receptor

The rat peripheral cannabinoid receptor (rCB2) was cloned from a Sprague-Dawley rat spleen cDNA library and when translated, encodes a protein of 410 amino acids. Alignment of rCB2 with mouse (mCB2) and human (hCB2) peripheral cannabinoid receptors reveals a high degree of homology except in the carboxy terminus where rCB2 is 50 and 63 residues longer than hCB2 and mCB2, respectively. PCR screening and sequencing of rat genomic DNA showed that rCB2 is encoded by three exons interrupted by two introns, one of which is polymorphic and contains a 209 base pair B2 (SINE) element. By Northern hybridization and ribonuclease protection assay (RPA), rCB2 mRNA was detected in rat spleen, testis, thymus and lung but not in rat brain, heart, kidney or liver. Like hCB2 and mCB2 receptors, rCB2 activates mitogen-activated protein kinase when it is stably expressed in Chinese Hamster Ovary (CHO) cells. The importance of the carboxy terminus in regulating CB2 receptor desensitization and internalization is well-established. Thus, the profound differences identified in this region of the CB2 receptor between species mandates caution when extrapolating experimental results from non-human models to the effects of chronic CB2 receptor stimulation in humans.     

Characterization of proteins that associate with an unglycosylated form of the transferrin receptor in A431 cells

A protein doublet (Mr = 135,000/130,000) was found to coprecipitate with an unglycosylated form of the transferrin receptor in tunicamycin-treated A431 cells. This doublet is not detected with either a monoclonal or polyclonal antibody to the transferrin receptor on Western blots indicating that these proteins do not interact directly with transferrin receptor antibody. Proteolytic digestion patterns of the individual proteins of the Mr = 135,000/130,000 doublet suggest that they are related to one another and are distinct from the transferrin receptor. Further characterization of these proteins indicates that they form a high molecular weight complex with the unglycosylated but not the glycosylated form of the transferrin receptor. Pulse-chase experiments demonstrate that the proteins post-translationally associate with the receptor.     

Immunolocalization and Biochemical Characterization of N-methyl-D-aspartate Receptor Subunit NR1 from Rat Brain

The N-methyl-D-aspartate (NMDA) receptor subunit NR1 gene can produce eight isoforms in rat brain. A novel methodology for purifying NMDA receptor NR1 subunit from rat brain is reported here using chicken polyclonal antibodies (IgYs) against synthetic peptides corresponding to N1, C1 and C2′ cassettes. The isolated protein was recognized by produced IgYs and commercial anti-NR1 IgGs, shown by MALDI-TOF MS a MW = 131,192 Da (glycosylated form); the enzymatically deglycosylated protein revealed a MW = 102,754 Da. The NMDA receptor NR1 subunit was characterized as being a heavily N-glycosylated protein. The isoelectric point was determined (6.3) as being different from that predicted for any of the isoforms (7.9–9.02). Attempts to separate the isoforms from the purified NR1 were unsuccessful, indicating the presence of just one isoform (NR1₁₁₁). Immunohistochemistry on hippocampus regions CA1, CA3 and Dentate gyrus with anti-N1, anti-N2 and anti-C2′ IgYs showed different staining intensity, depending upon the antibody assayed.     

Seizure Activity Produces Differential Changes in Adenosine A1 Receptors within Rat Hippocampus

Specific ligand binding to rat hippocampal adenosine A₁ receptor after administration of the convulsant drug 3-mercaptopropionic acid (MP) was studied by means of a quantitative autoradiographic method. 2-Chloro-N6-[cyclopentyl-2,3,4,5-³H adenosine] ([³H]CCPA), a potent and selective A₁ receptor ligand, was selected for binding studies. MP administration (150 mg/kg, i.p.), at seizure, caused significant increases in the following CA1 layers: pyramidal (45%), radiatum (18%) and lacunosum molecular (35%); in CA2 area, a significant decrease in stratum oriens (36%) and an increase in stratum radiatum (14%) and lacunosum molecular (33%) layers was observed. In CA3 area a rise in pyramidal (40%) and radiatum layers (26%), as well as in hillus (97%) was found. At postseizure, changes were restricted to CA1, CA2 and CA3 pyramidal layers and to CA1 lacunosum molecular layer, with increases ranging from 22 to 50%. These results show that [³H]CCPA binding is modified diversely in intrahippocampal layers and areas, thus indicating their dissimilar role in seizure activity.     

Modulation of ATP-induced LTP by cannabinoid receptors in rat hippocampus

Cannabinoids exert powerful action on various forms of synaptic plasticity. These retrograde messengers modulate GABA and glutamate release from presynaptic terminals by acting on presynaptic CB1 receptors. In particular, they inhibit long-term potentiation (LTP) elicited by electrical stimulation of excitatory pathways in rat hippocampus. Recently, LTP of the field excitatory postsynaptic potential (fEPSP) induced by exogenous ATP has been thoroughly explored. The present study demonstrates that cannabinoids inhibit ATP-induced LTP in hippocampal slices of rat. Administration of 10 μM of ATP led to strong inhibition of fEPSPs in CA1/CA3 hippocampal synapses. Within 40 min after ATP removal from bath solution, robust LTP was observed (fEPSP amplitude comprised 130.1 ± 3.8% of control, n = 10). This LTP never appeared when ATP was applied in addition to cannabinoid receptor agonist WIN55,212-2 (100 nM). Selective CB1 receptor antagonist, AM251 (500 nM), completely abolished this effect of WIN55,212-2. Our data indicate that like canonical LTP elicited by electrical stimulation, ATP-induced LTP is under control of CB1 receptors.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-012-9296-5) contains supplementary material, which is available to authorized users.     

Expression of pituitary adenylate cyclase-activating polypeptide and PACAP type 1 receptor in the rat gastric and colonic myenteric neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to regulate gastric acid secretion and intestinal motility. In the present study, the pattern of distribution of PACAP and PACAP type 1 receptor (PAC1) immunoreactivities were examined in the rat stomach and distal colon using a specific polyclonal antibody raised against rat/human PAC1. Western blot of the membrane preparations of NIH/3T3 cells transfected with the human PAC1 obtained by using rabbit polyclonal anti-PAC1 antibody showed a protein band with a molecular mass of approximately 50 kDa. NIH/3T3 cells transfected with the human PAC1 and incubated with the anti-PAC1 antibody displayed surface cell-type immunoreactivity, which was internalized following ligand exposure. In gastric or colonic longitudinal muscle/myenteric plexus (LMMP) whole mount preparations as well as cryostat sections, PACAP immunoreactivity was observed in cell bodies within the myenteric ganglia and nerve fibers in the muscle layers and mucosa. PAC1 immunoreactivity was confined mainly on the surface of the nerve cells. PACAP and PAC1 immunoreactivities showed a similar pattern of distribution in gastric and colonic tissues. Adjacent sections or LMMP whole mount preparations labeled with protein gene product 9.5 (PGP 9.5) revealed the neuronal identity of myenteric cells bearing PAC1. The neuronal localization of PACAP and PAC1 receptors supports their role in the neural regulation of gastric acid secretion and gastrointestinal motor function.     

Localization of vanilloid receptor 1 (TRPV1/VR1)-like immunoreactivity in goldfish and zebrafish retinas: Restriction to photoreceptor synaptic ribbons

The vanilloid receptor type 1 (TRPV1/VR1) is a non-specific calcium-permeable ionotropic cation channel expressed in the peripheral sensory system as well as in the central nervous system. An endogenous ligand for TRPV1 is arachidonoyl ethanolamide (anandamide), which also activates the metabotropic cannabinoid receptor 1 (CB1). Previous studies in this laboratory reported CB1 receptors and CB1-mediated effects on voltage-gated currents in goldfish cones and bipolar cells. In this study, we show TRPV1-like-immunoreactivity (TRPV1-L-IR) by immunoblot analysis of goldfish retina and rat brain homogenates with a guinea pig polyclonal antibody against the C-terminus of the rat TRPV1. Light-level immunocytochemistry showed restriction of the guinea pig-TRPV1 antibody to a very narrow band in the outer plexiform layer in goldfish and zebrafish retinas. However, no immunoreactivity was detected using rabbit-polyclonal antibodies against the C or N-termini of the rat TRPV1. Pre and post-embedding electron microscopy (EM) immunocytochemistry revealed that TRPV1-L-IR (using the guinea pig antibody) was restricted to synaptic ribbons of all cones and many rods, but never was observed at the synaptic ribbons of bipolar cells. While pre-embedded tissue showed diffuse label associated only with photoreceptor-synaptic ribbons, analysis of post-embedded tissue showed label tightly restricted to synaptic ribbons of all cones and many rods. Oblique sections showed that immunogold particles were confined to the outer electron dense region of the ribbons, with few or no gold particles in the ribbon core or associated with tethers or vesicles. Although TRPV1-L-IR described here, does not necessarily represent TRPV1 antigen associated with synaptic ribbons, these data provide an unequivocal label with which to study the functional dynamics of ribbon formation and degradation in teleost photoreceptors.     

Endocannabinoids Differentially Modulate Synaptic Plasticity in Rat Hippocampal CA1 Pyramidal Neurons

Background

Hippocampal CA1 pyramidal neurons receive two excitatory glutamatergic synaptic inputs: their most distal dendritic regions in the stratum lacunosum-moleculare (SLM) are innervated by the perforant path (PP), originating from layer III of the entorhinal cortex, while their more proximal regions of the apical dendrites in the stratum radiatum (SR) are innervated by the Schaffer-collaterals (SC), originating from hippocampal CA3 neurons. Endocannabinoids (eCBs) are naturally occurring mediators capable of modulating both GABAergic and glutamatergic synaptic transmission and plasticity via the CB1 receptor. Previous work on eCB modulation of excitatory synapses in the CA1 region largely focuses on the SC pathway. However, little information is available on whether and how eCBs modulate glutamatergic synaptic transmission and plasticity at PP synapses.

Methodology/Principal Findings

By employing somatic and dendritic patch-clamp recordings, Ca²⁺ uncaging, and immunostaining, we demonstrate that there are significant differences in low-frequency stimulation (LFS)- or DHPG-, an agonist of group I metabotropic glutamate receptors (mGluRs), induced long-term depression (LTD) of excitatory synaptic transmission between SC and PP synapses in the same pyramidal neurons. These differences are eliminated by pharmacological inhibition with selective CB1 receptor antagonists or genetic deletion of the CB1 receptor, indicating that these differences likely result from differential modulation via a CB1 receptor-dependent mechanism. We also revealed that depolarization-induced suppression of excitation (DSE), a form of short-term synaptic plasticity, and photolysis of caged Ca²⁺-induced suppression of Excitatory postsynaptic currents (EPSCs) were less at the PP than that at the SC. In addition, application of WIN55212 (WIN) induced a more pronounced inhibition of EPSCs at the SC when compared to that at the PP.

Conclusions/Significance

Our results suggest that CB1 dependent LTD and DSE are differentially expressed at the PP versus SC synapses in the same neurons, which may have an impact on synaptic scaling, integration and plasticity of hippocampal CA1 pyramidal neurons.     

Different molecular weight forms of opioid receptors revealed by polyclonal antibodies

Polyclonal antibodies were raised against a purified opioid receptor from bovine brain (Cho, et. al., 1986), and shown to inhibit 3H-diprenorphine binding to this receptor in a dose-dependent fashion. These antibodies were then used to characterize opioid-binding material present in rat brain and in NG108-15 neuroblastoma-glioma hybrid cells. Western blot analysis revealed that the antibodies reacted with a single species of 58,000 molecular weight in rat brain membranes; this closely corresponds in size to the bovine opioid receptor used to raise the antibodies. In contrast, the polyclonal antibodies reacted with a 45,000 molecular weight species in NG108-15 neuroblastoma-glioma hybrid cells; moreover, this band was specifically reduced in NG108-15 cells in which opioid receptors had been down-regulated by incubation with D-ala2-D-leu5-enkephalin for 24 hours. Thus at least two distinct opioid receptor molecules have been identified, which have antigenic similarities.     

Isolation and characterization of carbonic anhydrases from the marine diatom Phaeodactylum tricornutum

Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CA_ext) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO₂-grown cells. CA_ext was detected only in UTEX 640 grown under CO₂-limited conditions and present in trace amounts in cells grown on high CO₂. CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster-running band which was absent from extracts of high CO₂-grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CA_ext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low-CO₂-inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO₂-grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air-grown cells indicated that the CA_ext was located in the periplasmic space and on the cell membrane, whereas in high CO₂-grown cells only internal CA was detected.     

Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system.

The cannabinergic system is present in a variety of organs and tissues that perform a wide range of essential physiologic functions making it an inherently important therapeutic target for drug discovery. In order to augment our knowledge regarding the interactions between cannabinoid receptors (CBs) and their ligands, efficient and effective tools are essential for robust expression and purification of these membrane-bound proteins. In this report, we describe a suitable method for purification of the human cannabinoid receptor 2 (CB2) to a qualitative and quantitative level sufficient for mass spectral analysis. We utilized a baculovirus expression system, incorporating several epitope tags to facilitate purification and to ameliorate the effect the tags have on CB2 expression and function. Expressed protein encoded by a carboxy (C)-terminal His-tagged CB2 construct displayed a B(max) value of 9.3 pmol/mg with a K(D) of 7.30 nM using [3(H)]CP-55(940), a standard cannabinoid radioligand, and was selected for subsequent purification experiments. Western blot analysis of purified membrane protein yielded several forms of CB2, the most abundant being a 41 kDa peptide. A second protein species was observed with an apparent molecular weight of 46 kDa representing a glycosylated form of CB2. In addition, a CB2 homodimer was also identified. The purified receptor was subjected to mass spectroscopic analysis to confirm its identity and purity. Mass spectra corresponding to the intracellular, extracellular and transmembrane domains were obtained. These experiments exemplify the importance of high-level expression systems when developing membrane-bound protein purification strategies. This work will aid in the identification of receptor-ligand binding sites, the characterization of molecular features involved in receptor activation, and the elucidation of the CB2 receptor tertiary structure.