Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells.

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 +/- 0. 6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-alpha also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.

Local anesthetics inhibit the G protein-mediated modulation of K+ and Ca++ currents in anterior pituitary cells.

The effects of local anesthetics (LAs) on G protein-mediated responses of voltage-dependent K+ (I(K)) and Ca++ currents in rat anterior pituitary tumor (GH3) cells were analyzed by using a whole-cell voltage clamp. Extracellular lidocaine inhibited I(K) with an IC50 of 1.9 mM, comparable to 2.6 mM for I(Ba) but 10 times higher than the IC50 for I(Na) (0.17 mM). Low concentrations of lidocaine (30-100 microM), which had no direct effect on basal I(K), attenuated both the stimulatory and inhibitory modulation of K+ channels by thyrotropin-releasing hormone (TRH). Both modulations had an IC50 approximately 40 microM independent of [TRH]. Intracellular QX314 (100 microM), a quaternary, charged form of lidocaine, also significantly attenuated the TRH effects; however, external QX314 and the neutral LA benzocaine (100 microM) did not. Lidocaine (

Reoxygenation of hypoxic human umbilical vein endothelial cells activates the classic complement pathway.

BACKGROUND: Ischemia-reperfusion injury leads to the activation and endothelial deposition of complement. We investigated whether exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia and/or reoxygenation activates complement and decreases HUVEC-surface expression of the C3 regulatory proteins CD46 and CD55. METHODS AND RESULTS: HUVECs were subjected to 0, 12, or 24 hours of hypoxia (O2 = 1%) and then reoxygenated for 3 hours (O2 = 21%) in the presence of 30% human serum. C3 deposition and HUVEC-surface expression of CD46 and CD55 were evaluated by ELISA and flow cytometry. C3 deposition on HUVECs subjected to 12 or 24 hours of hypoxia followed by 3 hours of reoxygenation was significantly greater than normoxic HUVECs. Inhibition of the classic but not the alternative complement pathway during reoxygenation attenuated C3 deposition. Western blot analysis of HUVEC lysates under reducing conditions demonstrated significantly increased iC3b deposition in hypoxic/reoxygenated HUVECs compared with normoxic HUVECs. FACS analysis confirmed iC3b deposition. HUVEC-surface expression of CD46 and CD55 increases after hypoxia and/or reoxygenation. CONCLUSIONS: We conclude that (1) hypoxia and reoxygenation of HUVECs significantly increases iC3b deposition on HUVECs, (2) C3 deposition after hypoxia and reoxygenation is largely mediated by the classic complement pathway, and (3) HUVEC-surface expression of CD46 and CD55 increases after hypoxia and reoxygenation. These data demonstrate that hypoxia and reoxygenation of human endothelial cells activates the classic complement pathway despite an increase in complement C3 regulatory proteins.

Propofol and ethanol produce additive hypnotic and anesthetic effects in the mouse.

The sedative and anesthetic effects of ethanol and propofol when these drugs are coadministered are not known. Accordingly, we investigated the nature of the pharmacological interaction between ethanol and propofol during hypnosis and anesthesia in the mouse. Propofol, ethanol, and mixtures of the two were administered through the tail vein in male CD-1 mice (n = 162). The loss of righting response occurring 10 s after injection and persisting at least 10 s thereafter was defined as hypnosis, and lack of a motor response to tail clamping 60 s after injection was defined as anesthesia. The 50% effective dose (ED50) values for the hypnotic and anesthetic actions of the drugs were determined with quantal dose-response curves, using probit analysis. The pharmacological interactions were identified by the locations of ED50 values on their corresponding hypnosis and anesthesia isoboles. For each drug alone, the hypnotic and anesthetic ED50 values with 0.95 confidence intervals were 16.70 (11.98, 23.20) mg/kg and 25.02 (20.27, 31.29) mg/kg for propofol and 0.88 (0.81, 0.95) g/kg and 1.80 (1.45, 2.23) g/kg for ethanol, respectively. For the drugs in combination, the ED50 values for hypnosis with 0.95 confidence intervals were 6.98 (6.50, 7.49) mg/kg propofol with 0.61 (0.57, 0.66) g/kg ethanol, and for anesthesia were 10.55 (9.76, 11.42) mg/kg propofol with 0.93 (0.86, 1.05) g/kg ethanol, respectively. When plotted isobolographically, we found these combinations to be behaviorally additive both for hypnosis and anesthesia. Although a finding of synergism would have excluded the possibility of an identical mechanism of action for the drugs, elucidation of the molecular basis of the additivity must await further studies.

Estrogen-specific target site identified by progesterone-11 alpha-hemisuccinate-(2-[125I]-iodohistamine) in mouse brain membranes.

Using photoaffinity labeling with the progesterone analogue, progesterone-11 alpha-hemisuccinate-(2-[125I]-iodohistamine) ([125I]-his-PG), we identified and characterized a protein band of MW 29 kDa (p29) in mouse cerebellar membranes whose labeling is markedly inhibited by estrogens. Inhibition of the labeling was specific with respect to steroid structure. Labeling was strongly inhibited by estradiol-17 beta, estradiol-17 alpha, and the anti-estrogen tamoxifen and the synthetic estrogen diethylstilbestrol. Other estrogens (estriol, estrone) were less effective and the steroids, dihydroandrosterone, androsterone and aldosterone were ineffective. Preincubation with estradiol-17 alpha or estradiol-17 beta inhibited the labeling in a dose-dependent manner with IC50 values of 0.3 and 2.0 microM, respectively. The extent of labeling was three times as high in cerebellar membranes from males as from females. In males, labeling of cerebellar membranes was greater than that of cortex or limbic region. The labeling pattern of p29 was also different in membranes prepared from cerebellum, heart and liver. Moreover, PG enhanced the labeling of p29 in liver demonstrating a tissue-specific mode of interaction. The present results characterize p29 as a membrane-bound estrogen target site.

Pregnenolone sulfate increases intracellular Ca2+ levels in a pituitary cell line.

We have investigated the rapid steroid effects on intracellular calcium ([Ca2+]i levels in a clonal pituitary cell line (GH3). Among the steroids tested only pregnenolone sulfate induced a rapid and transient [Ca2+]i increase within 1 min. The specificity of pregnenolone sulfate-induced [Ca2+]i increase with respect to steroid structure was pronounced. Other steroids (5-40 microM) including pregnenolone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, progesterone, estradiol-17 beta, testosterone, 5 alpha-dihydrotestosterone, 5 alpha-dihydrotestosterone, 5 alpha-dihydroprogesterone, and 3 alpha,5 alpha-tetrahydroprogesterone were found to be ineffective. The [Ca2+]i increase with pregnenolone sulfate (30 microM) was completely abolished in a Ca(2+)-free medium or in the presence of La3+ (0.1 mM) and Co2+ (5 mM). The organic Ca2+ channel blockers methoxyverapamil (100 microM) and nicardipine (5 microM) both showed similar inhibitions (> 73%). The interaction between pregnenolone sulfate and voltage-gated Ca2+ channels (VGCC) was shown by coapplication of pregnenolone sulfate (10 microM) with Bay K 8644 (0.1 mM) or KCl (15 mM). Coapplication of pregnenolone sulfate with KCl increased the [Ca2+]i in an additive manner. However, with the specific agonist Bay K 8644(+/-), the pregnenolone sulfate effect was potentiated in a majority of the cells, suggesting cooperative interaction between the two. The results demonstrate that pregnenolone sulfate induces a rapid Ca2+ influx in GH3 cells. The marked nicardipine block also suggests that most of the Ca2+ influx is mediated through L-type VGCC.

Photoaffinity labeling with progesterone-11 alpha-hemisuccinate- (2-[125I]iodohistamine) identifies four protein bands in mouse brain membranes.

The radiolabeled progesterone (PG) analogue progesterone-11 alpha-hemisuccinate-(2-[125I]iodohistamine) was used to label PG binding proteins in brain membranes from mouse cerebellum. Photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified specific PG binding protein bands 1-4 of 64-29 kDa. Bands 1 and 4 were well resolved on the gel and easily quantified. Preincubation with PG inhibited photolabeling in a dose-dependent manner. The labeling was specific with respect to steroid structure. For band 1, the extent of inhibition of labeling by PG and 3 alpha, 5 alpha-pregnanolone (3 alpha) was pronounced. Other steroids such as testosterone (Tes), estradiol (Est), and corticosterone (Cor) were less effective, whereas pregnenolone sulfate (PS) and cholesterol (Cho) were ineffective. With respect to band 4, Est was the most effective; PG, 3 alpha, and Tes were intermediate; and PS, Cho, and Cor were ineffective. The results describe specific membrane proteins that bind PG (band 1) and Est (band 4).

Analgesia with anesthetic steroids and ethanol.

Ethanol is well known to enhance the potencies of a range of anesthetics. However, its effects on steroid anesthesia have not been reported. Because ethanol often is added to the delivery vehicle to increase steroid solubility, it is important to evaluate its contribution to steroid anesthesia. Intravenous injection of ethanol and anesthetic steroid(s) produced marked analgesia and a marked increase in the number of mice exhibiting the loss of righting response (LRR). Ethanol alone at these low doses was merely sedative, and steroid alone was hypnotic without any analgesic benefit. The nocifensive response to tail clamp was used as a measure of analgesia. Progesterone or the anesthetic steroid, 3 alpha-hydroxy-5 alpha- pregnan-20-one (3 alpha) alone reduced, but did not abolish the nocifensive response. Progesterone (80 mg/kg) or 3 alpha (3 mg/kg) administered together with ethanol (1.1 g/kg) abolished the nocifensive response (analgesia) and resulted in LRR within 10 s. The onset of both effects was rapid, and the duration was greater than 8 min. With 3 alpha alone, the reduced nocifensive behavior fully returned before the mouse regained the righting response. Thus, the evaluation of the nocifensive response is not precluded by LRR. The rapid onset of LRR and analgesia suggest that 3 alpha and progesterone, not their metabolites, are responsible for the observed activities.

3 alpha-hydroxy-5 alpha-pregnan-20-one is the only active anesthetic steroid in anesthetized mouse brain.

It was demonstrated that the anesthetic steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one mediates the loss of the righting response (LRR) in mice, in contrast to its metabolites, which are formed in vivo. To reach these conclusions, it was necessary to quantitate levels for 3 alpha-hydroxy-5 alpha-pregnan-20-one and its metabolites at the time of LRR. Methods were used that blocked the production of essentially all of the metabolites. Chromatography of brain extracts by thin-layer chromatography and high-performance liquid chromatography after anesthesia with 3 alpha-hydroxy-5 alpha-[3H]pregnan-20-one showed only a single peak that corresponded to 3 alpha-hydroxy-5 alpha-[3H]pregnan-20-one.

Mitochondrial phosphate transport. N-ethylmaleimide insensitivity correlates with absence of beef heart-like Cys42 from the Saccharomyces cerevisiae phosphate transport protein.

The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.