Regulatory sequences for the transcription of the laminin B2 gene in astrocytes.

Astrocytes synthesize only the B2 chain of laminin and that this chain is sufficient to stimulate neurite outgrowth. In this study, we have examined laminin B1 and B2 promoter constructs in various cell types in order to understand the transcriptional regulation of laminin B2 gene in astrocytes. Comparison of nuclear factor binding by Southwestern analysis with the highly active B2 promoter fragment revealed different patterns of nuclear factor binding. In HepG2 cells, two proteins of 105 and 98 kDa were identified while, in primary astrocytes, human U251 and rat C6 glioma cells, a greater number of nuclear proteins ranging from 43 to 212 kDa were detected. The laminin B1 promoter construct was inactive in transient transfection experiments in astrocytes yet active in the HepG2 hepatoma cells which synthesize both the B1 and B2 chains. In contrast, the laminin B2 promoter construct was active in both astrocytes and HepG2 cells. These results are consistent with the lack of laminin B1 mRNA expression in astrocytes and suggest that the differential regulation of the laminin B1 and B2 gene is controlled at the transcriptional level. Delineation of the 5'-flanking regions responsible for basal levels of B2 laminin promoter activity revealed a silencer-like segment between -830 and -224 which reduced promoter activity. Deletion analysis further revealed that B2 laminin promoter possesses a highly active short promoter (-94 to +106) and basal transcriptional activity resides within -61 to +106. DNase 1 footprinting, gel-shift competition assays and site-directed mutagenesis of a highly active short promoter revealed that this region contained binding sites for cell-type nuclear factors. The shortest construct containing only residues -21 to +106 was inactive in HepG2 and U251 glioma cells. In primary astrocytes, however, this construct showed a high level of transcriptional activity. Deletion of 47 bp (+59 to +106) in 5'-UTR completely blocked promoter activity in astrocytes confirming that this downstream region is important for transcriptional activity in primary astrocytes. Together, these results suggest that astrocytes may utilize mutually exclusive transcription factors and regulatory sequences, in addition to common factors in the control of the laminin B2 promoter.

In vivo comparison of cytochrome aa3 redox state and tissue PO2 in transient anoxia.

We have determined, in vivo and simultaneously, the tissue PO2 (Ptio2) and the oxidation-reduction (redox) state of cytochrome aa3 (cyt aa3) of cat cerebral cortex during and after a short period of N2 breathing. Thirteen cats were anesthetized, ventilated mechanically with room air, subjected to a limited bilateral craniotomy, and then injected with 25 mg/kg of pyrenebutyric acid (PBA) intravenously. Ptio2 was measured from PBA generated fluorescence, emitted by monitored cerebral cortical cells. The cyt aa3 redox state was measured from differential absorption of monochromatic light at 605 vs. 590 nm reflected from the same cortical cells. In response to a 1.5-min N2 ventilation (phase I) the increase in PBA fluorescence signal, indicating a decline in Ptio2, lagged behind the cyt aa3 reduction. When the animal was ventilated with room air (phase II), rapid reoxidation, followed by hyperoxidation of cyt aa3 occurred. The decrease in PBA fluorescence signal, indicating an increase in Ptio2, was seen to lag behind cyt aa3 reoxidation. These results indicate that hysteresis exists in the relation between Ptio2 and cyt aa3 redox state. This may be the result of the situation that 1) low tissue O2 concentration is partially compensated by accumulation of reduced cyt aa3, and 2) following brief periods of anoxia, the affinity of cyt aa3 to O2 is increased.

Enhancement of cortical evoked potentials by high atmospheric pressures of helium.

Adult guinea pigs with electrodes chronically implanted in the optic chiasm (OC(, lateral geniculate nucleus (LGN), and visual cortex (CX) were compressed at 1 bar/min to 120 bars pressure in helium-oxygen. Body temperature was controlled to within +/- 1 degree C, CO2 was removed by a soda-lime absorbent. Electrical stimuli (50 microA, 0.05 msec) were delivered to the OC at 9.6/sec at 10 bar intervals, with pressure held constant. Pressure caused only negligible changes inthe amplitudes of pre- and postsynaptic components of potentials in the LGN. Amplitudes of evoked potentials in the CX increased approximately linearly with pressure, reaching values of up to 300% above normobaric. Latencies did not change independently of temperature. Excitability of nerve fibers changed very little, but intracortical synaptic transmission was substantially enhanced. Differences in the effects of elevated helium pressure at the LGN and CX may be attributed to differences in organization of local circuits.

In vivo comparison of cerebral tissue PO2 and cytochrome aa3 reduction-oxidation state in cats during hemorrhagic shock.

To assess the adequacy of oxygen availability and utilization within the cerebral cortex in vivo, we have measured the partial pressure of oxygen in tissue (PtO2), as well as the reduction oxidation state of cytochrome c oxidase (cyt aa3) during shock induced by slow or rapid hemorrhage in anesthetized cats. PtO2 was measured with pyrenebutyric acid-generated fluorescence in cerebral cortical cells. Cyt aa3 redox state was measured by the absorption of monochromatic light at 605 nm absorption peak of the enzyme reflected from the same cortical field. The PtO2 remained within the normal range until either 30 +/- 1.5 ml blood/kg was removed or the mean arterial pressure fell by 70 +/- 5% of base line. Beyond either point, the PtO2 fell rapidly to a low value approximating zero. By contrast, the reduction of cyt aa3 began early when as little as 5 ml blood/kg was removed. Thereafter, the shift toward reduction was progressive and continuous with a slow rate at first and a rapid rate later. This accelerated rate of cyt aa3 reduction preceded the rapid fall of PtO2. We concluded that, under these experimental conditions, cyt aa3 reduction is a much earlier and more sensitive indicator of perturbed intracellular aerobic metabolism due to hemorrhage that is PtO2.

Correspondence of brain and rectal temperatures of guinea pigs in helium environments.

Brain and rectal temperatures were measured in guinea pigs exposed to helium-oxygen environments at pressures as high as 50 bars. Very high correlations (r = .856, P less than 0.001) between the two values were observed, with an average difference of 0.47 degrees C (computed SD = 0.31). Brain temperature was usually higher than rectal. At a given ambient temperature, heat loss at 50 bars was more rapid than at 20 bars, which supports existing data for lower pressures. At 50 bars, normal equilibrium temperatures (approximately equal to 39 degrees C) could be maintained only if ambient temperature was in the vicinity of 35 degrees C. Lower ambient temperatures resulted in lower equilibrium temperatures. Between 33 and 39 degrees C, brain temperature can be predicted from rectal temperature, with an error of about 0.5 degree C.

Redox transitions in mitochondria of cat cerebral cortex with seizures and hemorrhagic hypotension.

Fluorometry and dual-wave-length spectrophotometry were used to detect transitory shifts in the redox state of mitochondrial NADH and cytochrome aa3 in the exposed cerebral cortex of anesthetized paralyzed cats as seizures were induced with pentylenetetrazol. In normotensive animals, NADH and cytochrome aa3 oxidation accompany the seizures, but when the mean arterial pressure (MAP) is reduced to 40.2 +/- 1.1% of the base line by hemorrhaging, the NADH fluorescence response converts to a biphasic oxidation-reduction sequence. In extreme hypotension (MAP lowered to an average of 28%), only NAD reduction transients are observed with seizures, and cytochrome aa3 is oxidized irrespective of the low MAP. Our data show that a reversible perfusion impairment, perhaps inhomogeneous in its distribution, appears in the cortex at the 40% MAP level and modifies electron flux in the respiratory chain between NADH and cytochrome aa3, and uniform oxygen insufficiency is an unlikely cause for the reversal of NADH oxidation toward reduction during seizures under hypovolemic conditions.

Responses of retinal and visual pathway potentials of the guinea pig to nitrogen and helium at high pressure.

Electroretinographic, optic chiasm, and visual cortex potentials were monitored in the awake guinea pig as nitrogen pressures were raised to 16 ATA and held for 30 min. Pressurization to 90 ATA with helium in 10-ATA increments followed. We sought to (a) quantify the depressant effect of nitrogen on the retina as compared to the central visual pathway, and (b) to test for pressure reversibility. The electroretinogram was reduced approximately 15%, the chiasm potential by 15%, and the cortical response by 32% in 16 ATA nitrogen, and latent periods for the three signals increased an average of 5-8%. Helium at pressure did not restore the amplitude of the electroretinogram or optic chiasm response, but the visual cortex potential returned to control levels near 90 ATA total pressure. Reversal of the nitrogen-induced latency increases by helium was partially effective up to 40 ATA. Control latent periods were not reached. Further attenuation of the electroretinogram and chiasm response amplitudes, and increased latencies of all signals were observed at 50-90 ATA.

Oxygen tensions measured in cat cerebral cortex under hyperbaric conditions.

Pyrenebutyric acid (PBA), the intracellular fluorescent indicator, was used to measure the partial pressure of oxygen (PO2) in the exposed cerebral cortex of anesthetized cats at hyperbaric pressures up to 4 ATA. The validity of the PBA method for determining cortical PO2 was confirmed by demonstrating a precise linear relationship between Pao2 and the reciprocal of the fluorescence of PBA in the brain as the cat was ventilated with sequentially greater oxygen pressures while holding the Paco2 nearly constant. Increments in the Paco2 while the Pao2 was maintained at a high (about 2,000 Torr) level resulted in stepwise greater oxygen tensions in the brain until an oxygenation end point was reached with a Paco2 averaging near 122 Torr. Greater amounts of CO2 did not bring the mean PO2 of the brain, 1,017 Torr, closer to 2,000 Torr. During normocapnia the cortical PO2 was greater than the PO2 of cerebral venous blood collected from the superior sagittal sinus; however, in hypercapnia (PaCO greater than 45 Torr), the PO2 of the sinus blood exceeded the value determined in the cortex. This latter observation is taken as evidence for convective shunting of cerebral arterial blood to venous circulation when hypercapnia is present.

Oxidation of cerebral cytochrome aa3 by oxygen plus carbon dioxide at hyperbaric pressures.

The reduction-oxidation level of cytochrome aa3 in the intact cerebral cortex of cerveau isolé or pentobarbital-anesthetized cats was monitored by means of dual-beam reflectance spectrophotometry. Respiratory gases containing varying fractions of carbon dioxide and oxygen were administered at increased pressures, allowing titration of the cytochrome redox state from maximum reduction during nitrogen ventilation to the completely oxidized state. We show that the fully oxidized state could be reached with about 5% CO2-95% O2 inspired at 4 ATA. Maximum oxidation was achieved only through the relaxation of oxygen-induced vasoconstriction of the cerebral vessels, as our data indicated that large blood volume responses accompanied the increases in inspired carbon dioxide. With out technique, we have established that under resting air-breathing conditions cytochrome aa3 is about 85% reduced in the cat cerebral cortex, and that the absorption peak for cytochrome aa3 in situ is located near 602 nm. A free energy change of an additional 8 kcal is calculated to occur with donation of an electron pair from 85% reduced cytochrome aa3 to oxygen as compared to a 1% reduction level.