Localization of Endomorphin-2-Like Immunoreactivity in the Rat Medulla and Spinal Cord

Martin-Schild, S., J. E. Zadina, A. A. Gerall, S. Vigh and A. J. Kastin. Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord. Peptides 18(10) 1641–1649, 1997.—Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) are endogenous ligands that have greater affinity and selectivity for the μ-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin–biotin–peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing μ receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.     

Synthesis and binding activity of endomorphin-1 analogues containing beta-amino acids

Endomorphin-1 (Tyr-Pro-Trp-PheNH₂) has been proposed as the most potent endogenous ligand of the μ-opioid receptors. In this paper, we describe the synthesis of some endomorphin-1 based tetrapeptides in which a residue of the sequence Tyr-Pro-Trp-PheNH₂ is replaced by the corresponding β-isomer. These novel peptides showed different affinities for the opioid receptors labeled with [³H]-DAMGO in rat brain membranes, depending on the β-amino acid. In particular, the tetrapeptide containing β-Pro (Tyr-β-(R)-Pro-Trp-PheNH₂) displayed a higher affinity than endogenous endomorphin-1, as revealed by their K_i values (0.33 and 11.1 nM, respectively).     

Search of the human proteome for endomorphin-1 and endomorphin-2 precursor proteins

Based on the promising opioid pharmacological profile of the peptide, Tyr-Pro-Trp-Gly-NH(2) (Tyr-W-MIF), Zadina et al. [Zadina, J.E., Hackler, L., Ge, L.-J., Kastin, A.J., 1997. A potent and selective endogenous agonist for the mu-opiate receptor. Nature 386, 499-5502] synthesized and screened other Gly(4)-substituted peptides, culminating in the synthesis of Tyr-Pro-Trp-Phe-NH(2) (endomorphin-1), which displayed high affinity and selectivity for the mu-opioid receptor. The amidated peptide was then isolated from bovine brain frontal cortex, as was a related peptide, Tyr-Pro-Phe-Phe-NH(2) (endomorphin-2), that displayed similar high affinity and selectivity for the mu-opioid receptor. The biosynthesis of the endomorphins in the brain remains obscure, since the putative precursor proteins for the peptides have not been identified. With the completion of the human genome sequencing project, we hypothesized that we should uncover the biological precursors of the peptides using a bioinformatic approach to search the current human proteome for proteins that contained the endomorphin peptide sequences followed by Gly-Lys/Arg, the consensus sequence for peptide alpha-amidation and precursor cleavage. Twelve proteins were identified that contained the endomorphin-1 Tyr-Pro-Trp-Phe sequence, however none contained the Tyr-Pro-Trp-Phe-Gly sequence necessary for alpha-amidation. Twenty-two distinct proteins contained the endomorphin-2 tetrapeptide sequence, and two of those contained the sequence, Tyr-Pro-Phe-Phe-Gly, however, none contained the requisite peptide-Gly-Lys/Arg sequence. Western blot analysis using an endomorphin-2 antibody detected 4 prominent proteins in mouse brain, necessitating reinterpretation of previous immunocytolocalization studies in the brain. Screening of the current human proteome yielded no evidence for endomorphin precursor proteins based on accepted biochemical criteria.     

From MIF-1 to endomorphin: the Tyr-MIF-1 family of peptides

The Tyr-MIF-1 family of small peptides has served a prototypic role in the introduction of several novel concepts into the peptide field of research. MIF-1 (Pro-Leu-Gly-NH₂) was the first hypothalamic peptide shown to act “up” on the brain, not just “down” on the pituitary. In several situations, including clinical depression, MIF-1 exhibits an inverted U-shaped dose–response relationship in which increasing doses can result in decreasing effects. This tripeptide also can antagonize opiate actions, and the first report of such activity also correctly predicted the discovery of other endogenous antiopiate peptides. The tetrapeptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂) not only shows antiopiate activity, but also considerable selectivity for the mu-opiate binding site. Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH₂) is an even more selective ligand for the mu receptor, leading to the discovery of two more Tyr-Pro tetrapeptides that have the highest specificity and affinity for this site. These are the endomorphins: endomorphin-1 is Tyr-Pro-Trp-Phe-NH₂ and endomorphin-2 is Tyr-Pro-Phe-Phe-NH₂. Tyr-MIF-1 proved, contrary to the then prevailing dogma, that peptides can be saturably transported across the blood–brain barrier by a quantifiable transport system. Unexpectedly, the Tyr-MIF-1 transporter is shared with Met-enkephalin. In the era in which it was doubtful whether a peripheral peptide could exert CNS effects, the Tyr-MIF-1 family of peptides also explicitly showed that they can exert more than one central action that persists longer than their half-lives in blood. These peptides clearly illustrate that the name of a peptide restricts neither its actions nor its conceptual implications.     

Metabolism of Prostaglandin D2 by Human Cerebral Cortex into 9α, 11β-Prostaglandin F2 by an Active NADPH-Dependent 11-Ketoreductase

Abstract: In homogenates of rat cerebral neocortex prostaglandin D₂ (PGD₂) was found to be quantitatively the main PG biosynthesized by a cytosolic PGD synthetase from en-dogenously released arachidonic acid. Amounts of 628 ng/g wet weight were found after 30-min incubation periods compared with basal levels of 2.3 ng/g wet weight. In human cerebral cortex, whether obtained at biopsy or postmortem, only small amounts of PGD₂ (4.5–11.7 ng/g wet weight/30 min) were formed. Furthermore, PGD₂, added to homogenates of human biopsy temporal cortex, was converted efficiently into 9α,11β-PGF₂ by a NADPH-dependent 11-ke-toreductase as has been reported in other human tissues (liver and lung). PGF_2α was determined directly as the fl-butylbo-ronate derivative. It became clear that 9α,11β-PGF₂ was formed in considerably greater amounts than PGF_2α and that other metabolites are also formed. These results can account for the low amounts of PGD₂ found in incubations of human brain tissue. The rat brain does not contain 11-ketoreductase activity. The present results indicate that the 9α, 11β-PGF₂ must be considered along with other eicosanoids in pathophysiological situations in brain.     

Comparison of histamine-H1 receptors in bovine intracerebral microvessels with cerebral grey matter by [H]mepyramine binding

Small intracerebral blood vessels (microvessels) of bovine brain are known to contain the vasoactive amine histamine, and the presence of histamine-H₁ receptors in microvessels was examined using the radioligand, [³H]mepyramine. Microvessels were isolated from cerebral cortex grey matter, striatum and hippocampus by a sieving technique and membranes prepared for binding studies. [³H]Mepyramine bound to a single, high affinity site, which displayed stereoselectivity for (+) chlorpheniramine relative to its (−) isomer and was consistent with binding to H₁-receptors. The density of binding sites (B_max), in microvessel membranes from cortical grey matter, was approximately one-third of that seen in membranes prepared from cortical grey matter. Microvessels isolated from striata and hippocampi had a similar density of H₁-receptor sites to that seen in cortical microvessels.

These results demonstrate that bovine intracerebral microvessels contain significant numbers of histamine-H₁ receptors and strengthen the hypothesis that histamine could regulate the calibre of intracerebral blood vessels.     

Development of High Affinity Selective VIP1 Receptor Agonists

Gourlet, P., A. Vandermeers, P. Vertongen, J. Rathe, P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. Development of high affinity selective VIP₁ receptor agonists. Peptides 18(10) 1539–1545, 1997.—The biological effects of VIP are mediated by at least two VIP receptors: the VIP₁ and the VIP₂ receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP₁ receptor selective agonists derived from secretin and GRF. [R¹⁶]chicken secretin had IC₅₀ values of binding of 1, 10,000, 20, and 3000 nM for the rat VIP₁-, VIP₂-, secretin- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP₁ receptor (IC₅₀ of 60 nM). The chimeric, substituted peptide [K¹⁵,R¹⁶,L²⁷]VIP(1-7)/GRF(8-27) had IC₅₀ values of binding of 1, 10,000, 10,000 and 30,000 nM for the rat VIP₁-, VIP₂-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC₅₀ of 0.8 nM for the human VIP₁ receptor and a low affinity for the human VIP₂ receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

Effects of JO 1784, A selective sigma ligand, on the autoradiographic localization of M2 and M2 muscarinic receptor subtypes in trimethyltin treated rats

The distribution patterns of M₁ and M₂ muscarinic receptor subtypes following TMT and JO 1784 administration in the male Sprague-Dawley rat were investigated. In the present study, JO 1784 was injected in doses of 1, 4 and 16 mg/kg i.p. for one week prior to the single injection of TMT (8 mg/kg i.p.) and subsequently for 33 days. The effects of JO 1784 on the density of muscarinic receptor sub-types (M₁ and M₂) in the control and trimethyltin (TMT) treated rats were then evaluated. The topographic distribution and changes in muscarinic (M₁ and M₂) receptor densities were determined by means of autoradiography using [³H]quinuclidinylbenzilate (QNB). Both sub-types of muscarinic receptors contributed to the observed decrease in total muscarinic receptor binding in TMT-treated rats. In control rats, JO 1784 alone decreased M₁ receptor density in the amygdaloid nuclei, basal ganglia, cortex and hippocampus and decreased M₂ receptor density in the amygdaloid nuclei, basal ganglia, cortex, hippocampus, hypothalamus and septal regions. In TMT treated rats, chronic JO 1784 administration has a “neuroprotective effect” on both M₁ and M₂ receptors subtypes. Thus, following chronic administration of JO 1784 to TMT treated rats, both increases and decreases in M₁ receptor density were observed relative to TMT animals. A significant increase in M₁ receptor density was found in the cortex, olfactory regions, septum, thalamus and basal forebrain nuclei. In the hippocampus (CA₂ and CA₃), a significant decrease in M₁ receptor density was observed. In TMT-treated rats, JO 1784 produced a significant increase in M₂ receptor density in several brain regions with the most marked effects occurring in the amygdaloid nuclei, basal ganglia, cortex, hippocampus and hypothalamus. The ability of the selective sigma ligand, JO 1784, to attenuate the loss of muscarinic receptors in TMT treated rats could be of importance in the development of novel neuroprotective drugs.     

Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2

Previously, we have shown that substitution of Pro² for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [³H][(1S,2R)ACPC²]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [³H][(1S,2R)ACPC²]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K_d = 1.80 ± 0.21 nM and receptor density, B_max = 345 ± 27 fmol × mg protein⁻¹ at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na⁺ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [³H][(1S,2R)ACPC²]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.     

Autoradiographic Visualization of the Receptor Subclasses for Vasoactive Intestinal Polypeptide (VIP) in Rat Brain

Vertongen, P., S. N. Schiffmann, P. Gourlet and P. Robberecht. Autoradiographic visualization of the receptor subclasses for Vasoactive Intestinal Polypeptide (VIP) in rat brain. Peptides 18(10) 1547–1554, 1997.—Vasoactive Intestinal Polypeptide (VIP) exerts its biological effects through interaction with two high affinity receptors named the VIP₁- and the VIP₂ receptors. Their messenger RNAs have been mapped in rat brain by in situ hybridization. A cyclic peptide (RO 25-1553) and a secretin analogue ([R¹⁶]chicken secretin) were identified as selective agonist peptides for the VIP₂- and VIP₁ receptors, respectively. The iodinated peptides retained the high affinity and selectivity of the unlabelled peptides and were used for the mapping of each receptor subclass in rat brain. VIP₁ receptors were present in the cerebral cortex, the piriform cortex, the claustrum, the caudate-putamen, the dentate gyrus, the lateral amygdaloïd nucleus, the anteroventral thalamic nucleus, the rhomboïd nucleus, the supraoptic nucleus and the choroïd plexus. VIP₂ receptors were present in the cerebral cortex, the claustrum, the caudate-putamen, the nucleus accumbens, the lateral septal nuclei, the bed nucleus of the stria terminalis, the basolateral amygdaloïd nucleus, the Ammon’s horn, the thalamic nuclei except some centromedial nuclei, the medial habenula, the suprachiasmatic nucleus, the periventricular nucleus, the mammilary nucleus, the superior colliculus and the choroïd plexus.     

Immunochemical comparison of pertussis toxin substrates in brain and peripheral tissues

The tissue distribution of pertussis toxin-sensitive GTP-binding proteins was examined using specific antibodies raised against the purified -subunit of G₀ from bovine brain or against synthetic peptides predicted from cDNAs for distinct G_i subtypes. GTP-binding proteins were partially purified from membrane fractions prepared from rabbit tissues including brain, heart, liver, lung, erythrocytes and neutrophilis. Brain contained both G₀ and G₁. G_il was also found to be abundant in heart. All peripheral tissues contained tissues contained readily detectable amounts of G₁₂, whereas only barely detectable amounts of G_i2 were found in brain. G_i3 was found to be prominent in erythorocytes and exists as a minor component of G proteins in neutrophils and liver. Thus, G_i2 appears to be widely disseminated in peripheral rabbit tissues, while other pertussis toxin substrates are more limited in their distribution.     

[H]WAY–100635 for 5–HT1A receptor autoradiography in human brain: a comparison with [H]8–OH–DPAT and demonstration of increased binding in the frontal cortex in schizophrenia

WAY–100635 is the first selective, silent 5–HT_1A (5-hydroxytryptamine_1A, serotonin-1A) receptor antagonist. We have investigated the use of [³H]WAY–100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5–HT_1A receptor agonist, [³H]8–OH–DPAT. Saturation studies showed an average Kd for [³H]WAY–100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [³H]WAY–100635 binding and [³H]8–OH–DPAT binding were similar. The density of [³H]WAY–100635 binding sites was 60–70% more than that of [³H]8–OH–DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [³H]WAY–100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [³H]WAY–100635, we confirmed an increase of 5–HT_1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [³H]8–OH–DPAT. Compared to [³H]8–OH–DPAT, [³H]WAY–100635 has two advantages: it has a higher selectivity and affinity for the 5–HT_1A receptor, and it recognizes 5–HT_1A receptors whether or not they are coupled to a G-protein, whereas [³H]8–OH–DPAT primarily detects coupled receptors. Given these considerations, the [³H]WAY–100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [³H]8–OH–DPAT binding seen in the same cases is attributable to an increase of 5–HT_1A receptors rather than any other binding site. Second, the enhanced [³H]8–OH–DPAT binding in schizophrenia reflects an increased density of 5–HT_1A receptors, not an increased percentage of 5–HT_1A receptors which are G-protein-coupled. We conclude that [³H]WAY–100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5–HT_1A receptors in the human brain.     

Expression of serotonin receptor mRNAs in blood vessels

Using RT-PCR we distinguished mRNAs for all known G-protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1 _β, 5-HT_2A, 5-HT_2B, 5-HT₄, and 5-Ht₇ receptor mRNA to different extents. New splice variants of the porcine 5-HT₄ receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5-HT_{1 β}, 5-HT_2b, and 5-HT₄ receptor mRNA, whereas in smooth muscle cells 5-HT_{1 β}, 5-HT_2A, 5-HT₇, and in some experiments 5-HT_2B receptor mRNA were found. A model for the regulation of vascular tone by different 5-HT receptors is proposed.     

Receptor binding profiles of NPY analogues and fragments in different tissues and cell lines

To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4–25-fold higher affinity for the Y₂ receptor than for the Y₁ receptor. The affinity of [Leu³¹,Pro³⁴]NPY is 7–60-fold higher for the Y₁ receptor when compared with the Y₂ subtype. Species selectivity within the Y₂ receptors is demonstrated by PYY(3–36), NPY(2–36), NPY(22–36), and NPY(26–36). It is shown that NPY(22–36) is species selective for the human Y₂ subtype (K_i of 0.3 nM) compared with the rabbit and rat Y₂ receptor (K_i of 2 and 10 nM, respectively). PYY(3–36) displays highest affinity for the human and rabbit Y₂ subtype (K_i of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y₂ receptor is shown by NPY(2–36) and PYY(3–36). In addition, PYY(3–36) and NPY(2–36) are not only subtype selective, but also species selective.     

In Vitro Properties of a High Affinity Selective Antagonist of the VIP1 Receptor

Gourlet, P., P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. In vitro properties of a high affinity selective antagonist of the VIP₁ receptor. Peptides 18(10) 1555–1560, 1997.—A selective high affinity VIP₁ receptor antagonist [Acetyl-His¹, D-Phe², Lys¹⁵, Arg¹⁶, Leu¹⁷] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP₁ receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP₁-, rat VIP₂- or the human VIP₂- recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP₁ receptor. The IC₅₀ values of ¹²⁵I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP₁-, rat VIP₂-, human VIP₁- and human VIP₂ receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP₁ receptor mediated stimulation of adenylate cyclase activity. The K_i values were respectively of 15 ± 5 nM and 2 ± 1 nM for the rat and human VIP₁ receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP₁ receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.     

Does zaltoprofen antagonize the bradykinin receptors?

Zaltoprofen is a nonsteroidal antiinflammatory drug that has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B₂ receptor. In order to test the predictive power of this claim, we applied the drug to vascular smooth muscle assays previously found useful to characterize B₂ receptor antagonists (contractility, human isolated umbilical vein) or B₁ receptor antagonists (contraction, rabbit aorta; relaxation, rabbit mesenteric artery). Zaltoprofen (up to 30 μM) failed to antagonize BK or des-Arg⁹-BK-induced contraction in the umbilical vein and aorta, respectively. The drug (1 μM) abated des-Arg⁹-BK-induced, prostaglandin-mediated relaxation of the precontracted mesenteric artery, consistent with its known activity as a cyclooxygenase (COX) inhibitor. However, zaltoprofen (10 μM) did not inhibit kinin-stimulated phospholipase A₂ activity in HEK 293 cells expressing recombinant forms of the rabbit B₁ or B₂ receptors. Nonpeptide antagonists of either receptor subtype were active in this respect. The results do not support that zaltoprofen, a COX inhibitor, antagonizes kinin receptors or influences their signaling with selectivity in the tested systems.     

GABA(A) receptor beta(2) subunit mRNA content is differentially regulated in ethanol-dependent DBA/2J and C57BL/6J mice

Chronic ethanol treatment is known to alter gene expression and function of γ-aminobutyric acid type-A (GABA_A) receptors. Here we focus on the β₂ subunit which is widely expressed in the mammalian brain, and plays a key role in the GABA binding site. Previous studies using rodent models of ethanol dependence show either increased or no change of β₂ subunit mRNA and peptide content following chronic ethanol administration. In humans, polymorphism at the β₂ subunit is associated with ethanol dependence in some, but not all, populations. In the present study we measured mRNA content in the cerebellum and cerebral cortex using ethanol-naive and ethanol-dependent DBA/2J and C57BL/6J mice. The DBA/2J strain displays severe ethanol withdrawal severity, while the C57BL/6J strain shows milder withdrawal reactions. RNase protection analysis demonstrated that the DBA/2J strain is more sensitive to ethanol-induced increases in β₂ subunit mRNA content in the cerebellum, showing significant increases at lower blood ethanol concentrations than C57BL/6J mice. The ethanol-induced regulation in C57BL/6J mice appears to be more complex, with decreases in β₂ subunit mRNA content at low blood ethanol concentrations, and increases at higher concentrations. These data suggest that differences between C57BL/6J and DBA/2J mice in the degree of physical dependence (withdrawal) on ethanol may be related to differential sensitivity to ethanol regulation of β₂ subunit expression.     

Identical properties of transmembrane synaptic vesicle protein Mr 100,000 in Torpedo and Mr 86,000 in bovine brain

Synaptic vesicles derived from the Torpedo electric organ and bovine cerebral cortex contain concanavalin A binding transmembrane glycoproteins of M_r 100,000 and 86,000, respectively. Their isolelectric points range from 5.5 to 6.0. On deglycosilation both glycoproteins yield identical products of M_r 62,000. The fully glycosilated and the deglycosilated proteins from both Torpedo and bovine brain are recognized by the monoclonal anti-SV2 antibody (Buckley and Kelly, J. Cell Biol. 100, 1284–1294, 1985) as well as by a monospecific IgG fraction raised against Torpedo vesicles and immunopurified against the bovine brain M_r 86,000 glycoprotein. This is shown by Western blotting as well as by immunoaffinity isolation with one antibody and immunodetection with the other antibody. Furthermore on immunohistochemical analysis of the Torpedo electric organ both antibodies recognize exactly the same nerve terminal ramifications. It is concluded that the glycoproteins of M_r 100,000 in Torpedo and of M_r 86,000 in bovine brain are corresponding proteins with different degrees of glycosilation.     

Diphosphatidylglycerol-induced changes in the organization of mitochondrial ATPase

1. On submitochondrial particles from bovine heart, diphosphatidylglycerol produced a selective solubilization of ATPase. The solubilized enzyme was purified further by ammonium sulfate fractionation and shown to have the same reconstitutive activity as coupling factor F₁ (Pullman, M.E., Penefsky, H. S., Datta, A. and Racker, E. (1960) J. Biol. Chem. 235, 3322–3329).

2. Diphosphatidylglycerol-treated submitochondrial particles retained large amounts of the phospholipid and showed a decreased ATPase activity. Once the excess of phospholipid was removed, soluble ATPase could be again reincorporated in an oligomycin-sensitive complex.

3. On Mg-ATP particles the solubilization of ATPase induced by diphosphatidylglycerol was preceded by a stimulation of oligomycin-sensitive ATPase which indicated a dissociation of F₁ from the ATPase inhibitor (Pullman, M. E. and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762–3769). Magnesium was required to obtain the oligomycin-sensitive stimulation whereas in the absence of magnesium the solubilization of ATPase was prevalent.

4. It is concluded that the decreased association of F₁ with the ATPase inhibitor produced by diphosphatidylglycerol, causes a labilization of ATPase-membrane interaction. Under particular conditions, e.g. a high amount of phospholipid and a low concentration of magnesium, this is followed by the detachment of ATPase.