Effects of acute hypoxia on the adenylate cyclase and evans blue transport of brain microvessels

Kinetic parameters of the albumin transport were measured during and after an acute hypoxic insult evoked in newborn piglets by experimental bilateral pneumothorax. Adenylate cyclase activity was determined in the cerebral microvessels isolated by ultracentrifugation from different stages of brain damage. A decrease of the adenylate cyclase activity was observed in the cerebral microvessels of animals with acute hypoxic condition. However, the adenylate cyclase activity was found to be increased significantly in the microvessels during recirculation.

The activation of adenylate cyclase in the microvessel wall may be of pathogenetic importance in the development of vasogenic brain oedema.     

Treatment of cardiac myocytes with 8-(4-chlorophenylthio)-adenosine 3'',5''-cyclic monophosphate, forskolin or cholera toxin does not stimulate cellular or heparin-releasable lipoprotein lipase activities.

Incubation of isolated cardiac myocytes with 500 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) or 100 microM-forskolin for 2 1/2 h did not increase the heparin-induced release of lipoprotein lipase (LPL) into the medium. When LPL activity in cardiac myocytes was depleted by treatment of rats with cycloheximide (2 mg/kg; 2.5 h) and inclusion of the protein-synthesis inhibitor in the isolation solutions, incubation with CPT-cAMP or forskolin did not influence the rate of repletion of LPL activity in cells or the recovery of heparin-releasable LPL activity. Although the administration of cholera toxin (0.5 mg/kg; 16-17 h) to rats increased LPL activity in a low-speed supernatant fraction from heparin-perfused hearts, LPL activity was not increased in cardiac myocytes from cholera-toxin-treated rat hearts, and the heparin-induced release of LPL was unchanged. Incubation of cultured ventricular myocytes with 1 microgram of cholera toxin/ml or 500 microM-CPT-cAMP for 24 h did not increase cellular LPL activity or LPL released into the culture medium after a 40 min incubation with heparin. Therefore interventions that stimulate adenylate cyclase activity (forskolin, cholera toxin) or incubation with CPT-cAMP do not increase cellular LPL activity or promote the translocation of LPL to a heparin-releasable fraction in cardiac myocytes.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

In vivo definition of the functional origin of replication (ori(+ )) of the promiscuous plasmid pLS1

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the –10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.     

Calcium/Calmodulin-Stimulated Protein Kinase II Is Present in Primary Cultures of Cerebral Endothelial Cells

Abstract: Calcium/calmodulin-stimulated protein kinase II (CaMPK II). a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long-term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the α-subunit of CaM-PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that α-CaM-PK II can act as one of the key enzymes of calcium-mediated intracellular signaling in the cerebral endothelial cells and suggest that α-CaM-PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.