Ethanol enhances the in situ phosphorylation of MARCKS and protein kinase C activity in primary cultures of astrocytes.

Protein kinase C (PKC) plays an important regulatory role in astrocyte function. Chronic exposure to ethanol for 4 days resulted in an increase in Ca2+-dependent PKC activity in the supernatant fraction of astrocyte homogenates. Only Ca2+-independent PKC activity could be observed in the membrane fraction and this activity was unaffected by ethanol exposure. Chronic ethanol exposure also increased the in situ phosphorylation of MARCKS in permeabilized astrocytes both in the absence or presence of the PKC activator, phorbol 12 -myristate 13 -acetate (PMA). These results suggest an increase in the expression of one or more astrocytic PKC isoforms after chronic ethanol exposure.

Effects of human alpha-thrombin and 8bromo-cAMP on large and microvessel endothelial monolayer equivalent "pore" radii.

Previous studies have reported that endothelial cells isolated from large vessels compared with microvessels from the same or distinct organs showed considerable phenotypic and biochemical heterogeneity. In the present study we extend these findings by comparison of the effects of 8-bromo-cyclic adenosine monophosphate (8Br-cAMP), human alpha-thrombin, 8Br-cAMP followed 5 min later by thrombin or no treatment (control) on the equivalent "pore" radii (rp) of endothelial monolayers isolated from the main bovine pulmonary artery (BPAEC) compared with lung microvessels (BLMV). BLMV, isolated from a 1-cm peripheral segment of the lung, were significantly larger than those obtained from large vessels (1602 +/- 142 microns2 vs 398 +/- microns2, respectively). In addition, BLMV monolayers formed a heteroporous barrier with less size-selectivity compared with BPAEC monolayers. 8Br-cAMP caused monolayers of both cell types to close their large "pores" which completely restricted the passage of solute molecular radii > 35-60 A across these barriers, consistent with a rp of approximately 75-100 A. This effect was due to a reduction in the area available for solute exchange (Ap) and/or an increase in the path length of the transport pathway (delta X). Human alpha-thrombin produced an increase in the Ap/delta X consistent with the formation of large open areas between adjacent cells that exposed the approximately 2000 A pore radius of the filter support. Since this effect was more marked in microvessel compared with large vessel monolayers, microvessel endothelial cells appear to be more sensitive to the effects of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of involvement of protein kinase C in ethanol-induced inhibition of metabotropic-glutamate receptor function in primary cultures of astrocytes.

The stimulation of [3H] inositol phosphate (InP) formation by the selective metabotropic-glutamate receptor agonist, 1S, 3R-ACPD, was significantly reduced in rat cortical astrocytes chronically exposed to 100 mM ethanol for 4 days. Under the same conditions, chronic ethanol either increased or did not affect the InP responses to norepinephrine and carbachol, respectively. The InP responses to all three agonists were sensitive to phorbol 12-myristate 13-acetate. Although the protein kinase C inhibitors, calphostin C and staurosporine, significantly relieved the ethanol induced inhibtion of the InP response to 1S, 3R-ACPD, these responses were still significantly less than corresponding values obtained from control cells treated with these inhibitors. The data suggests that mechanisms in addition to protein kinase C are responsible for the ethanol induced inhibition of metabotropic-glutamate function.

Pichinde virus-induced respiratory failure due to obstruction of the small airways: structure and function.

Respiratory distress that leads to death is seen in patients with Lassa fever. The development of this respiratory problem was studied using a Pichinde virus model (10(4) plaque forming units, IP, survival time 20 +/- 1 days) in strain 13 guinea pigs (n = 35, 229-353 g) of this lethal human contagious infectious disease. Extravascular lung water to bloodless dry lung weight (EVLW/BDLW) ratio showed a modest yet significant increase in animals 13 and 18-21 days postinoculation (PI). In contrast, residual lung blood and lung radioactive 125I-labeled human serum albumin activity index were elevated only in the 18- to 21-day group. These data are consistent with the progressive severity of perivascular edema, lymphocytic pneumonitis, and some alveolar protein between days 13 and 18-21 PI. Lymphocytic pneumonitis appeared to be distributed near most airways and was proportional to the degree of Pichinde virus antigen staining of alveolar macrophages, large mononuclear cells within the pulmonary vascular and extravascular spaces, and alveolar-capillary membranes. These findings suggest that lymphocyte recruitment to the lung reflects the Pichinde virus-induced cell-mediated immune response. Obstructed small bronchi with some lumenal cell debris and hypertrophied epithelial cells were found associated with the areas of marked pneumonitis. The severe hypoxemia and modest anaerobic metabolism in association with marked tachypnea and normocapnia are consistent with small airway obstruction and wasted ventilation, since no change in arterial blood pressure, heart rate, hematocrit, hemoglobin, or blood volume was noted. These data suggest that Pichinde virus-induced respiratory failure was due to obstruction of the small airways with wasted ventilation in association with lymphocytic pneumonitis.

Thrombin and bradykinin initiate discrete endothelial solute permeability mechanisms.

This study documents the discrete solute permeability mechanisms associated with physiologically high concentrations of human alpha-thrombin and bradykinin stimulation of bovine pulmonary artery endothelial cell (BPAEC) monolayers using fluorescein isothiocyanate-hydroxyethyl starch macromolecules. Agonist-induced alterations of intracellular free calcium ([Ca2+]i) using fura-2 acetoxymethyl ester were also measured. BPAEC monolayers showed restricted diffusion consistent with a small-pore (approximately 150 A) radius under baseline conditions. Thrombin produced a major increase in monolayer permeability that was greatest for solute molecular radii (ae) > 100 A. This effect was associated with the exposure of the large (approximately 2,000 A) pores of the filter support by 50- to 1,050-microns2 open areas between approximately 0.5% of the adjacent endothelial cells. This heterogeneous endothelial barrier of parallel large- and small-pore transport pathways permitted solute convection with free diffusion across a few large pores to dominate the restricted diffusion of most apparently unperturbed endothelial junctions. Bradykinin produced a small, transient elevation in monolayer permeability to ae < 35 A, consistent with an increase in the number of small pores or a decrease in path length of this transport pathway. The bradykinin- and thrombin-induced peak elevations in [Ca2+]i were inversely associated with the degree of increased monolayer solute permeability, and enzymatically inhibited thrombin produced none of these effects. These data show that bradykinin and human alpha-thrombin represent two distinct classes of endothelial cell agonists that initiate discrete solute permeability mechanisms.

Death after Pichinde virus infection in large and small strain 13 guinea pigs.

The lethal events of Pichinide virus infections were studied in large (> 36-week-old), small (4- to 6-week-old), and uninfected strain 13 guinea pigs. Time to death was shorter and the rate of body weight loss greater for large than for small virus-infected guinea pigs. Severely ill large guinea pigs developed a life-threatening, nonlactate metabolic acidemia with an increased anion gap, hypokalemia, and renal failure. Small infected guinea pigs near death demonstrated a modest increase in blood and plasma volume indices and in lung and heart permeability to albumin. This group showed a severe hypoxemia, elevated blood lactate, and bicarbonate ion concentrations with a doubled respiratory rate. These findings are consistent with respiratory failure due to obstruction of small airways. Although virus-infected guinea pig endothelial cells showed a decrease in DNA and protein synthesis, no change in bradykinin-induced intracellular calcium signaling was noted.

Restricted diffusion of macromolecules by endothelial monolayers and small-pore filters.

We studied the size-selective permeability and restricted diffusion (Rd) properties of macromolecules across bovine pulmonary artery endothelial cell (BPAEC) or epithelial (LLC-PK1) monolayers grown on polycarbonate (PC) filter supports using fluorescein isothiocyanate-hydroxyethyl starch (FITC-HES, 16 A less than ae less than 180 A). Most BPAEC seeded at subconfluent density and grown for 3-6 days produced barriers with marked Rd. This characteristic was similar to that measured across PC filters with pore radii (rp) of 150 and 250 A without cells. Rd of LLC-PK1 monolayers was consistent with a transport pathway rp of much less than 75A. BPAEC monolayers prepared by supraconfluent seeding showed convective solute transport due to a significant number of incompletely formed intracellular junctions. Most monolayers grown to confluence, or a thin layer of collagen type I prevented this effect, Rd was enhanced when BPAEC monolalyers were grown on this collagen network. These data suggest that the subendothelial layers, which includes basal lamina, pericyte, and interstitial collagen, may act as series resistors with the endothelium to provide the Rd observed in the microvascular wall in vivo. This may explain why BPAEC monolayers grown to confluence without subendothelial layers in vitro showed Rd consistent with large (150-250 A) rp that was significantly greater than those modeled as the small (approximately 50 A) "pore" or 6-A fiber matrix of the in vivo capillary wall.

Macromolecular transport across endothelial monolayers.

We studied the macromolecular size-selective transport characteristics of polycarbonate (PC) filters with defined pore radius (rp; 15,000 to 400 A) as well as 2,000 A rp PC filter-bovine pulmonary artery endothelial cell (EC) monolayer sandwich under zero hydrostatic pressure conditions using fluorescein isothiocyanate-hydroxyethyl starch (FITC-HES, 16 A less than ae less than 100 A), and 2-methoxy-2,4-diphenyl-3(2H) furanone-bovine serum albumin (MDPF-BSA, ae = 35.5 A). We surprised to find substantial convective solute transport (solute drag) across the filter-endothelial sandwich. This effect was increased by large rp (15,000 A) filters and prevented by 400 A rp filters. Positive hydrostatic pressure across 2,000 A rp filters increased convective solute transport and negative pressure prevented this effect. High, medium and low permeability monolayers on 2,000 A filters progressively attenuated the solute drag effect seen across these filters without cells. The decline in monolayer permeability was associated with an increased filter area covered by cells; approximately 50 and 95% as well as greater than 99%, respectively. Although significant restricted diffusion was seen across low permeability monolayers, this pattern was distinct from that measured in single frog capillaries. Restricted diffusion by low permeability monolayers under conditions that produce solute drag document the significant barrier effects of high confluence endothelial monolayers, in vitro. These data show that solute transport across endothelial monolayers is due to diffusion+convective solute drag. The degree of the solute drag effect across the filter-endothelial sandwich is a direct function of monolayer confluence.