Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of Microtubule-Associated Protein 2 and Brain Spectrin by Calpain: A Comparative Study

The in vitro degradation of microtubule-associated protein 2 (MAP-2) and spectrin by the calcium-dependent neutral protease calpain was studied. Five major results are reported. First, MAP-2 isolated from twice-cycled microtubules (2 X MT MAP-2) was extremely sensitive to calpain-induced hydrolysis. Even at an enzyme-to-substrate ratio (wt/wt) of 1:200, 2 X MT MAP-2 was significantly degraded by calpain. Second, MAP-2 purified from the total brain heat-stable fraction (total MAP-2) was significantly more resistant to calpain-induced hydrolysis compared with 2 X MT MAP-2. Third, MAP-2a and MAP-2b were proteolyzed similarly by calpain, although some relative resistance of MAP-2b was observed. Fourth, the presence of calmodulin significantly increased the extent of calpain-induced hydrolysis of the alpha-subunit of spectrin. Fifth, the two neuronal isoforms of brain spectrin (240/235 and 240/235E, referred to as alpha/beta N and alpha/beta E, respectively) showed different sensitivities to calpain. alpha N-spectrin was significantly more sensitive to calpain-induced degradation compared to alpha E-spectrin. Among other things, these results suggest a role for the calpain-induced degradation of MAP-2, as well as spectrin, in such physiological processes as alterations in synaptic efficacy, dendritic remodeling, and in pathological processes associated with neurodegeneration.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

High Expression of the HNK-1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.     

Suppression of 5α-androstane-3β, 17β-diol hydroxylase activity in rat pituitary by cobalt protoporphyrin: Implications for testosterone homeostasis

Cobalt protoporphyrin (CoPP) administered subcutaneously to adult male rats caused a marked reduction in the conversion of 5α-androstane-3β-17β-diol (3β-adiol) to its main triol derivative (6α-atriol) by homogenates of the pituitary but not of the prostate or brain (ventromedial hypothalamus and cortex). No effect in the brain was observed when this heme analogue was infused intracerebroventricularly. 3β-adiol hydroxylase, the enzyme responsible for the reaction and whose main function is thought to be the elimination of dihydrotestosterone and its metabolites from target tissues, was also inhibited by CoPP and SKF-525A added in vitro. The reaction was microsomal and dependent on NADPH. It is proposed that the lack of reciprocal elevation of luteinizing hormone in the face of the low testosterone levels observed following treatment with CoPP may be due, in part, to increased levels of androstanediols. These metabolites accumulate because of increased production from testosterone and decreased conversion to their triol derivatives in the pituitary.     

High-level expression and secretion of a lysine-containing analog of Escherichia coli heat-stable enterotoxin'

The mechanism of action of the heat-stable enterotoxin STa secreted from enterotoxigenic forms of Escherichia coli has remained elusive, in part due to a tedious, low-yield purification procedure. We report here a method for obtaining large amounts of a biologically active lysine-containing analog of STa. Initial attempts to express the toxin using an expression vector that did not encode a signal sequence resulted in no biologically active material being recovered either from lysed cells or as a secretory product. However, use of the secretion vector pJAL36, which contains the STII enterotoxin signal sequence, allowed large amounts of an STa derivative containing the additional sequence Ser-Thr-Lys at the amino terminus of the mature enterotoxin to be readily purified from culture supernatants. This enterotoxin analog, known as KSTa-1, was equal in biological and receptor binding activity to the native toxin STa. The lysine residue present in KSTa-1 promises to be useful as a reactive amino acid that is readily derivatized to allow coupling of the enterotoxin to supports for affinity chromatography and antigenic conjugates. Additionally, the insertion of the lysine residue carboxy terminal to the Ser-Thr sequence adds a reversible “handle” to the toxin sequence in that the Ser-Thr-Lys segment can be removed by treatment with trypsin, releasing the native form of STa.     

Physical violence,child abuse,and child homicide

The study of child abuse and child homicide has been based on the often implicit assumption that there is a continuum of violence ranging from mild physical punishment to severe abuse and homicide. Empirical data supporting this assumption are sparse. Existing data can be shown, however, to support an assumption that there are distinct forms of violence, not a continuum. This paper reviews these data and discusses their implications for the study of violence, abuse, and homicide in terms of substantive and methodological explanations. In addition, the implications of the assumption that violence consists of distinct behaviors as opposed to a continuum are discussed in light of sociobiological and evolutionary explanations of child abuse and child homicide. This paper was written under the auspices of the Family Violence Research Program at the University of Rhode Island. A complete list of books and articles is available upon request. Richard J. Gelles is Professor of Sociology and Anthropology and the Director of the Family Violence Research Program at the University of Rhode Island. He is the author or coauthor of 14 books and more than 90 articles and chapters on family violence. His most recent books areIntimate Violence, published in 1988 by Simon and Schuster;Physical Violence in American Families: Risk Factors and Adaptations in 8,145 Families, published by Transaction Books in 1990; andIntimate Violence in Families, published in 1990 by Sage Publications.     

Tissue distribution,metabolism, and elimination of perfluorooctanoic acid in male and female rats

The elimination, tissue distribution, and metabolism of [1-¹⁴C]perfluorooctanoic acid (PFOA) was examined in male and female rats for 28 days after a single ip dose (9.4 μmol/kg, 4 mg/kg). A sex difference in urinary elimination of PFOA-derived ¹⁴C was observed. Female rats eliminated PFOA-derived radioactivity rapidly in the urine with 91% of the dose being excreted in the first 24 hr. In the same period, male rats eliminated only 6% of the administered ¹⁴C in the urine. The sex-related difference in urinary elimination resulted in the observed difference in the whole-body elimination half-life (t_1/2) of PFOA in males (t_1/2 = 15 days) and females (t_1/2 < 1 day). Analysis of PFOA-derived ¹⁴C in tissues showed that the liver and plasma of male rats and the liver, plasma, and kidney of female rats were the primary tissues of distribution. The relatively high concentration of PFOA in the male liver was further examined using an in situ nonrecirculating liver perfusion technique. It was shown that 11% of the PFOA infused was extracted by the liver in a single pass. The ability of the liver to eliminate PFOA into bile was examined in rats whose renal pedicles were ligated to alleviate sex differences in the urinary excretion of PFOA. In a 6-hr period following IP administration of PFOA, there was no apparent difference in biliary excretion, where both males and females eliminated less than 1% of the PFOA dose via this route. We hypothesized that the sex difference in the persistence of PFOA was due to a more rapid formation of a PFOA-containing lipid (i.e., a PFOA-containing mono-, di-, or triacylglycerol, cholesteryl ester, methyl ester, or phospholipid) in the male rat. Also, the increased urinary elimination of PFOA in females may have been due to increased metabolism to a PFOA-glucuronide or sulfate ester. However, no evidence that PFOA is conjugated to form a persistent hybrid lipid was obtained, nor were polar metabolites of PFOA in urine or bile detected. In addition, daily urinary excretion of fluoride in male and female rats before or after PFOA treatment were similar, suggesting that the parent compound is not defluorinated. Thus, the more rapid elimination of PFOA from female rats is not due to formation of a PFOA metabolite.