The central cannabinoid receptor (CB1) mediates inhibition of nitric oxide production by rat microglial cells.

Upon activation, brain microglial cells release proinflammatory mediators, such as nitric oxide (NO), which may play an important role in the central nervous system antibacterial, antiviral, and antitumor activities. However, excessive release of NO has been postulated to elicit immune-mediated neurodegenerative inflammatory processes and to cause brain injury. In the present study, the effect of cannabinoids on the release of NO from endotoxin/cytokine-activated rat cortical microglial cells was evaluated. A drug dose-dependent (0.1 microM-8 microM) inhibition of NO release from rat microglial cells was exerted by the cannabinoid receptor high-affinity binding enantiomer (-)-CP55940. In contrast, a minimal inhibitory effect was exerted by the lower affinity binding paired enantiomer (+)-CP56667. Pretreatment of microglial cells with the Galphai/Galphao protein inactivator pertussis toxin, cyclic AMP reconstitution with the cell-permeable analog dibutyryl-cAMP, or treatment of cells with the Galphas activator cholera toxin, resulted in reversal of the (-)-CP55940-mediated inhibition of NO release. A similar reversal in (-)-CP55940-mediated inhibition of NO release was effected when microglial cells were pretreated with the central cannabinoid receptor (CB1) selective antagonist SR141716A. Mutagenic reverse transcription-polymerase chain reaction, Western immunoblot assay using a CB1 receptor amine terminal domain-specific antibody, and cellular colocalization of CB1 and the microglial marker Griffonia simplicifolia isolectin B4 confirmed the expression of the CB1 receptor in rat microglial cells. Collectively, these results indicate a functional linkage between the CB1 receptor and cannabinoid-mediated inhibition of NO production by rat microglial cells.

Collagen subtype pattern in normal and varicose saphenous veins in humans.

Our present study was to determine the collagen subtype pattern in the greater saphenous vein of the lower limb, obtained from 21 normal (macroscopically and ultrastructurally non-varicose vein segments from non-varicose subjects) and 37 varicose subjects, and to compare affected (macroscopically and ultrastructurally varicose segments from varicose veins) vs. non-affected (macroscopically and ultrastructurally non-varicose segments from varicose veins) segments (16). After elastase pretreatment and partial pepsin digestion, types I, III & V collagens (CI, CIII, CV) were extracted selectively by differential salt precipitation and measured quantitatively in samples obtained from normal and varicose saphenous veins-either affected or unaffected segments. Significant elevations of water (p < 0.05) and collagen type I [CI] (p < 0.01) content in varicose veins (both affected and unaffected segments) as compared with normal saphenous veins were observed. The collagen type III (CIII) and collagen type V (CV) content of varicose veins were found to be slightly reduced as compared to normal veins and consequently the CI/(CIII+CV) ratio in varicose veins increased significantly (p < 0.02) as compared to normal veins. Elevation of the CI/(CIII+CV) ratio in varicose veins may cause considerable weakening of the venous wall, further supporting the "weak wall" theory of varicose vein etiology.

Therapeutic effects of estradiol benzoate on development of collagen-induced arthritis (CIA) in the Lewis rat are mediated via suppression of the humoral response against denatured collagen type II (CII).

The effects of estradiol benzoate (EB) on the development of anti-CII antibodies and their pathogenic potential were studied during the progress of established CIA in the rat. CIA was induced in mature female Lewis rats by two subcutaneous inoculations containing bovine native CII (BCIIn), emulsified in Freund's incomplete adjuvant. Clinical arthritis fully developed by day 18 and then EB (1 mg/kg body wt per day, diluted in corn oil (CO)) was administered intramuscularly every second day thereafter. Antibodies binding four different CIIs (bovine or rat, either native or heat-denatured) were detected in sera and joint tissue extracts by means of solid-phase ELISA. Pharmacological doses of EB (>0.2 mg/kg body wt per day) caused significant remission of established CIA 5-7 days after treatment, and selectively suppressed the production of antibodies specific for denatured CII. To evaluate the arthritogenic potential of circulating anti-CIId IgG, transfer experiments were performed. IgG anti-CIIn, purified from EB-treated CIA rats, was not arthritogenic, whereas IgG anti-denatured (CIId), purified from CO-treated CIA rats, caused severe passive arthritis. Furthermore, pretreatment with rat CIId protected against subsequent induction of CIA, and this protection was associated with suppressed antibody production against CIId. Collectively, our results indicate that antibodies specific for CIId are involved in the pathogenesis of CIA, and that oestrogen-related remission of clinical arthritis may by caused by a selective suppression of antibodies produced against degraded/denatured CII.

Granulocyte-macrophage colony-stimulating factor enhances cationic antimicrobial protein synthesis by human neutrophils.

We analyzed the effect of recombinant human granulocyte macrophage CSF (GM-CSF) on protein synthesis in peripheral blood polymorphonuclear leukocytes. GM-CSF enhanced PMN 35S-methionine incorporation 1.5-fold over a 2-h incubation period. The effect of GM-CSF on the synthesis of specific proteins was investigated by separating the radiolabeled proteins on SDS-PAGE followed by autoradiography. Stimulation of protein synthesis by GM-CSF was rapid (2 h), dose dependent, and affected at least 10 separate polypeptides. Using ion exchange chromatography some of the GM-CSF-enhanced proteins were identified to have a cationic nature, including the 37- and the 57-kDA proteins whose synthesis was increased by GM-CSF 2.4-fold and 1.6-fold, respectively. The cationic protein fractions from GM-CSF-primed and control cells were eluted from an ion exchange column and tested for their antimicrobial activity. An overall twofold increase in the amount of cationic proteins was recovered from GM-CSF-treated cells as compared to control cells, and these proteins showed a proportional enhanced killing of S. typhimurium. These results suggest that enhanced cationic protein synthesis is an important mechanism whereby GM-CSF can increase neutrophil microbicidal activity via nonoxidative pathways.

Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor.

Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.