Evidence for tissue-associated α2-magcroglobulin in mouse skeletal muscle

α₂-Magcroglobulin (α₂M), a major serum protease inhibitor, was localized in mouse skeletal muscle by immunoperoxidase histochemistry. In all muscles examined (mm. soleus, plantaris, and extensor digitorum longus) specific immunoreactivity occurred diffusely in extracellular structures (periendomysium, blood vessel wall) as well as inside about a half of the muscle fibers. This localization pattern did not change substantially by extensively perfusing deeply anesthetized mice with phosphate buffered saline (PBS) to remove serum α₂M. In release experiments on fresh (nonfixed) cryostat sections, specific immunoreactivity persisted after an extensive prewash with PBS (up to 5–6h), but a new specific staining appeared inside those fibers that were originally negative. Western blotting experiments were negative on the soluble fraction of muscle homogenate, thus confirming that the perfusion procedure was effective in removing serum α₂M. By contrast, three specific bands (185, 165, and 35 kDa) appeared in detergent-solubilized extracts (0.3% Triton X-100), indicating the occurrence of tissue-associated α₂M. Confocal immunofluorescence microscopy revealed that the intracellular specific staining was associated to a longitudinal network, probably corresponding to the sarcoplasmic reticulum. A multifunctional role of α₂M in skeletal muscle was hypothesized.     

Differences in expression of GABAA receptor subunits, but not benzodiazepine binding, in the chick brainstem auditory system

Neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL) of the chick brainstem auditory system show an unusual physiological response to GABA. Examination of these nuclei usingin situ hybridization for GABA_A receptor subunits showed a differential expression of the γ₂ and α₁ subunits. The γ₂ subunit was found in both NM and NL, but the α₁ subunit was found in NL only. Like NL, other areas of the tissue section that showed labeling with the γ₂ probe, such as the medial vestibular nucleus (VeM) and granule cells of the cerebellum (CB), also labeled with the α₁ probe. Thus, given that NM labeled with the γ₂ probe, the absence of the α₁ subunit was unusual in this tissue. This difference in subunit composition suggests that there may also be a difference in GABA receptor function in NM compared to these neighboring areas. One feature of the GABA_A receptor believed to be related to the presence of γ₂ and α₁ subunits is specific pharmacological properties of the benzodiazepine modulatory site. It has been proposed that the α₁ subunit is necessary for producing a GABA_A receptor with a benzodiazepine site that has Type I binding characteristics. The present experiments challenge this hypothesis. Based on the differential presence of the α₁ subunit, one would expect that GABA receptors in NM would show different benzodiazepine binding properties than NL, VeM, and CB. However, displacement of³H-flunitrazepam binding using CL 218,872, which differentiates between the Type I and Type II receptors, showed no difference between these areas. Additionally, the relatively high affinity for CL 218,872 suggests that even NM contains Type I receptors.     

Serum macroglobulin induces prion protein transition

The accumulation of the protease-resistant (PrPRes) abnormal form of the cellular prion protein (PrPC) plays a central role in Transmissible Spongiform Encephalopathies. In this report we describe a blood serum protein, which is precipitated by 9% PEG-6000, migrates in a sucrose gradient with a density of 1.21–1.17 g/ml, has a molecular mass of approximately 720 kDa and amino acid sequence as α₂-Macroglobulin (α₂-M). This protein can potentiate the conversion of the human recombinant prion protein (23–231 AA) as well as PrPC from the brain of mouse, hamster or cow from the sensitive to the resistant form. Under physiological conditions (pH 7.5; 0.15 M NaCl; 37°C for 30 min; a nanomolar concentration range of PrP) the small, highly structured RNA (shs RNA) may activate the conversion process. We describe the design of an assay for screening for the biomolecules that prevent the PrP transition. Based on our experimental data we discuss the role of the α-helix to β-sheet conformational change in the PrPSen to PrPRes conversion as well as the role of the α₂-M during the initial stage of amyloidogenesis as well as the shs RNAs involvement in the formation of amyloid aggregates in neurodegenerative diseases. The text was submitted by the authors in English.     

Expression of α<Subscript>2</Subscript>-macroglobulin,neutrophil elastase,and interleukin-1α differs in early-stage and late-stage atherosclerotic lesions in the arteries of the circle of Willis

Different types of atherosclerotic (AS) lesions can be distinguished histologically and represent different stages of AS plaque development. Late-stage lesions more frequently develop complications such as plaque rupture and thrombosis with vessel occlusion than early AS lesions. To clarify whether protective, destructive, and inflammatory proteins are differentially expressed in early-stage and late-stage AS plaques we examined the proteinase inhibitor α₂-macroglobulin (A2M), the neutrophil elastase (NE)—an enzyme degrading elastin and collagen fibers—and the proinflammatory protein interleukin-1α (IL-1α) in all types of AS plaques in the arteries of the circle of Willis from 78 human autopsy cases of both genders (61–91 years of age). Paraffin sections of AS plaques were immunostained with antibodies directed against A2M, NE and IL-1α. In initial AS lesions A2M was found, whereas NE and IL-1α were absent. NE and IL-1α became detectable as soon as a significant number of macrophages occurred within AS lesions. With increasing histopathological type of AS lesions, a marked increase of the area of the plaque exhibiting NE and IL-1α was observed. The area which exhibits A2M in AS plaques, on the other hand, did not vary significantly between the different stages. Thus, our results indicate a disproportionately high increase of the destructive enzyme NE and the proinflammatory protein IL-1α in relation to A2M with the progression of the grade of AS lesions pointing to the transgression of the protective capacity of A2M by NE and IL-1α in late-stage plaques. Therefore, our findings support the hypothesis that NE-induced tissue damage in late-stage AS plaques contributes to the development of plaque rupture and subsequent thrombosis.     

Subunit- and brain region-specific reduction of GABAA receptor subunit mRNAs during chronic treatment of rats with diazepam

The mRNA levels for several GABA_A receptor subunits were measured by Northern blot analysis. Rats were treated for 3 wk by continuous release of diazepam (DZP) from subcutaneous reservoirs, and then sacrificed immediately or 48 h after removing the reservoirs. Poly(A)⁺ RNAs, isolated from cerebral cortex, cerebellum, and hippocampus, were hybridized with oligonucleotide probes for GABA_A receptor subunits and a cDNA probe for β-actin. Subunit mRNAs were expressed relative to the corresponding β-actin mRNA. DZP treatment decreased the α₁ subunit mRNA level 40% in hippocampus, but it was not changed in cortex or cerebellum. The α₅ subunit mRNA level was decreased in cerebral cortex (28%) and hippocampus (15%). The γ₂ subunit mRNA level was decreased (40%) only in cortex. DZP treatment did not affect α₂, α₃, α₄, β₂, or β₃ subunit mRNA levels. Decreases in mRNA levels had reversed within 48 h after stopping chronic treatment. Acute DZP did not change α₁, α₅, or γ₂ subunit mRNA levels. The decreases in GABA_A receptor subunit mRNA levels were specific to subunit and brain region. These results, coupled with those after chronic flurazepam treatment, also indicated that the effects on GABA_A receptor subunit mRNA levels are specific to the benzodiazepine (BZ) used for chronic treatment.     

Human astroglial but not microglial cells synthesize α2 in vitroin vitro

Alpha₂-macroglobulin (α₂ M) is a serum proteinase inhibitor with a broad specificity. At present its role in human brain is unknown, but recent data report its presence in the CNS, particularly at glial level. Previous studies from our group demonstrated the synthesis and secretion of α₂ M in different glial cultures derived from an astrocytoma and a glioblastoma. In the present study a human fetal astroglial cell line and two microglial established cell lines are examined for the presence of α₂ M by using polyclonal antibodies in ELISA and immunofluorescence assays. While we observed a strong specific positivity in the cytoplasm and in the culture medium of the GFAP, vimentine positive cells, no positivity was detected in FcR, Iysozyme positive microglial cells. Since interaction of proteinases and proteinase inhibitors appear to play a crucial role in the development of neuroimmunological compentence, these data suggest a dissociation of macro and micro-glia immune functions. Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

Distribution of Lys-γ2-melanocyte-stimulating hormone-(Lys-γ2-MSH)-like immunoreactivity in neuronal elements in the brain and peripheral tissues of the rat

Using an antiserum raised against Lys-γ₂-melanocyte-stimulating hormone (Lys-γ₂-MSH), with a high specificity for this peptide and its des-Lys derivative, γ₂-MSH, we found Lys-γ₂-MSH-like immunoreactivity to have a widespread distribution in the rat brain. In colchicine-treated rats, groups of immunopositive cell bodies were found in the intermediate and anterior lobes of the pituitary gland, in the hypothalamic arcuate nucleus and in the commissural part of the nucleus of the solitary tract (NTS). Immunopositive fibers were found to originate from the latter two cell body regions. The distribution of these fibers was similar to that of the pro-opiomelanocortin-containing cell bodies and projections as it has been described previously. Immunopositive terminals were found in brain regions containing neurons which have been shown to express mRNA for melanocortin receptors, though the distribution of Lys-γ₂-MSH-like immunoreactivity is considerably more widespread than that of mRNA for the ‘γ-MSH receptor’ (the melanocortin MC₃ receptor), which has been reported to be mainly expressed in the hypothalamus. In the periphery Lys-γ₂-MSH immunoreactivity was localized in the adrenal medulla and in neuronal fibers and varicosities in the heart. The vascular system, the bronchi and kidney were immunonegative. The occurrence of Lys-γ₂-MSH immunoreactivity in many of the brain regions which are involved in cardiovascular regulation offers leads for further studies on the putative role of γ-MSHs in cardiovascular control. The occurrence in the rat heart of Lys-γ₂-MSH-containing fibers suggests a role of the γ-MSHs in cardiac function.     

Functional coupling of GABAB receptors with G proteins that are sensitive to N-ethylmaleimide treatment, suramin, and benzalkonium chloride in rat cerebral cortical membranes

Summary. Although it is known that GABA_B receptors are negatively coupled to adenylyl cyclase, the detailed selectivity of functional interaction between GABA_B receptors and G_i subfamily members is still ambiguous. (±)-Baclofen-stimulated high-affinity GTPase activity, which was competitively antagonized by 2-hydroxy-saclofen, was attenuated by pretreatment of the membranes with N-ethylmaleimide (NEM) in a concentration- and incubation period-dependent manner. The NEM-pretreated (50 μM, 15 min at 4°C) membranes restored the (±)-baclofen-sensitive high-affinity GTPase activity when reconstituted with pertussis toxin-sensitive bovine brain G proteins. Among recombinant rat G_α subunits, G_iα−2 appeared most effective as compared with other subunits (G_iα−2 > G_iα−3 > G_iα−1 = G_oα). The GABA_B receptor-mediated high-affinity GTPase activity was also completely eliminated by 100 μM suramin and by 100 μM benzalkonium chloride. These results indicate that GABA_B receptors in rat cerebral cortex couple to NEM-sensitive G proteins, in particular G_i2, which are sensitive to suramin and benzalkonium chloride. Received September 6, 1999; accepted March 6, 2000     

Arachidonic acid and prostaglandin D2 cooperatively accelerate desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscles

To clarify the effects of arachidonic acid (AA) and its metabolites on desensitization of nicotinic acetylcholine (ACh) receptor channel in mouse skeletal muscle cells, we investigated the time-dependent decrease in the channel opening frequency of ACh (1 μM)-activated channel currents by the cell-attached patch clamp technique. AA (30–100 μM) applied to a patched membrane or to non-patched membrane accelerated the decrease in the channel opening frequency. A cyclooxygenase inhibitor, indomethacin (10 μM), prevented the acceleration elicited by 30 μM AA, but not by 100 μM AA. A lipoxygenase inhibitor, nordihydroguaiaretic acid (10 μM), and a cytochrome P-450 inhibitor, ketoconazole (3 μM), did not affect the acceleration by 30 μM AA. Prostaglandin (PG) D₂ at 10 μM alone and at 25 nM in combination with 10 μM AA accelerated the decrease in the channel opening frequency. No acceleration was observed with PGE₂ at 10 μM alone and at 25 nM in combination with 10 μM AA. Pretreatment with a protein kinase (PK) C inhibitor, staurosporine (10 nM), but not with a PKA inhibitor, H-89 (3 μM), prevented the acceleration elicited by AA+PGD₂. These results suggest that AA, and PGD₂ of its metabolites, cooperatively accelerate desensitization of nicotinic ACh receptor channel. The activation of PKC by AA and PGD₂ may be involved in the mechanism of the cooperative acceleration of desensitization.     

α-Synuclein immunoreactivity in dementia with Lewy bodies: morphological staging and comparison with ubiquitin immunostaining

α-Synuclein is a presynaptic protein recently identified as a specific component of Lewy bodies (LB) and Lewy neurites. The aim of this study was to assess the morphology and distribution of α-synuclein immunoreactivity in cases of dementia with LB (DLB), and to compare α-synuclein with ubiquitin immunostaining. We examined substantia nigra, paralimbic regions (entorhinal cortex, cingulate gyrus, insula and hippocampus), and neocortex (frontal and occipital association cortices) with double α-synuclein and ubiquitin immunostaining in 25 cases meeting neuropathological criteria for DLB. α-Synuclein immunostaining was more specific than ubiquitin immunostaining in that it differentiated LB from globose tangles. It was also slightly more sensitive, staining 4–5% more intracytoplasmic structures, especially diffuse α-synuclein deposits that were ubiquitin negative. In addition to LB, α-synuclein staining showed filiform and globose neurites in the substantia nigra, CA2–3 regions of the hippocampus, and entorhinal cortex. A spectrum of α-synuclein staining was seen in substantia nigra: from diffuse “cloud-like” inclusions to aggregated intracytoplasmic inclusions with variable ubiquitin staining to classic LB. We hypothesize that these represent different stages in LB formation. Received: 18 May 1999 / Revised: 28 July 1999 / Accepted: 30 July 1999