Scintigraphic evaluation of bacterial translocation during hemorrhagic shock.

We investigated the use of gamma scintigraphy to evaluate the temporal and spatial patterns of translocation of radiolabeled Escherichia coli from the porcine jejunum during and following hemorrhagic shock. Thirteen healthy mixed breed pigs (22-43 kg) were randomly allocated to two groups. Pigs were anesthetized with sodium pentobarital (30 mg/kg) and mechanically ventilated (100% O2). Each pig was instrumented for mean arterial pressure (MAP) and superior mesenteric artery (SMA) blood flow determination. A 25-cm loop of vascularly intact distal jejunum was isolated, and 10 mCi (10(11) cfu) of radiolabeled E. coli (99mTcO4-) was placed within the bowel segment. Consecutive 5-min scintigrams of the entire abdomen and thorax were collected for 6 hr. Pigs in the shock group (n = 7) were hemorrhaged such that MAP was maintained at 50-60 mm Hg for 5 hr. Pigs in the sham group (n = 6) were maintained without hemorrhage for 6 hr. The total radioactive counts in the translocation regions of the scintigram were plotted against time, and the slope of the regression lines was compared between groups. In the shock group, SMA blood flow decreased significantly (P < 0.05) during the hypotensive period but returned above baseline during reperfusion. The mean (+/- SD) slopes for translocation regression lines were 9.3 +/- 11.4 counts/min and 36.3 +/- 33.7 counts/min in the sham and shock groups, respectively (P < 0.05). Translocation was scintigraphically evident 50-100 min following induction of hemorrhage and did not require reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Spin-labeling studies of the conformation of the Ca(2+)-regulatory protein calmodulin in solution and bound to the membrane skeleton in erythrocyte ghosts: implications to transmembrane signaling.

Electron paramagnetic resonance (EPR) studies of the Ca(2+)-regulatory protein calmodulin (CaM) have been performed. The conformation of CaM in solution changes upon binding of Ca2+ allowing the protein to bind to target proteins existing in the red blood cell membrane. In this study a maleimide spin label, covalently attached to the single cysteine residue of CaM located in the first Ca(2+)-binding domain, was used to monitor allosteric conformational changes induced by interaction of CaM with Ca2+ and subsequently with the red blood cell membrane. The results show, relative to apo-CaM, a significant increase in the apparent rotational correlation time, tau, of the spin label when Ca2+ was present in solution (P less than 0.001). When apo-CaM exposed to red blood cell membrane ghosts in the absence of Ca2+, no significant difference in spin label motion was seen relative to solution, consistent with the idea that Ca2+ is required for CaM to bind to skeletal proteins. When Ca2+ was added to CaM which was then exposed to ghosts, a highly significant increase in tau (decrease in motion) (P less than 0.000001) relative to apo-CaM exposed to ghosts was found. This latter increase in tau is significantly greater than that produced by the addition of Ca2+ to CaM in solution (P less than 0.001). The major interaction sites of CaM were found by photoaffinity labeling and autoradiography on SDS-PAGE to be on the principal skeletal protein, spectrin. EPR was also used to investigate the biophysical correlates of transmembrane signaling. Spin-labeled CaM was bound to the membrane skeleton in the presence of Ca2+. On the opposite side of the erythrocyte membrane a lectin was bound to the external glycoconjugate of Band 3, the major transmembrane protein of the erythrocyte. A highly significant increase in T of the maleimide spin probe was found relative to the control system in which the lectin was absent. (P < 0.00001). These results suggest that electron paramagnetic resonance spectra of spin-labeled CaM can provide useful information about protein structure and function when in solution and when bound to membranes.

Spin labeling studies of wheat germ calmodulin in solution.

Electron paramagnetic resonance was used to investigate the physical state of plant calmodulin in solution. Wheat germ calmodulin contains a single cysteine residue (Cys-27) on the first of four calcium binding loops. In this study the nitroxide spin label 2,2,6,6-tetramethyl-4-maleimidopiperidine-1-oxyl (MAL-6) was covalently attached to Cys-27 to produce a Ca(2+)-sensitive, biologically-active, labeled protein. The rotational correlation time of the spin label, a measure of its rotational mobility and reflective of the physical state of this region of the protein, was calculated under various conditions. Relative to control, changes in the physical state of the protein reflected by increased motion of the spin label were observed at high pH, low ionic strength and upon addition of Ca2+. These results extend knowledge of the structure of the protein, previously known from solid state and biochemical studies, to calmodulin in solution.

Water transport across red-cell membranes following reductive methylation of the major transmembrane protein, Band 3: implications to increased divalent cation membrane permeability.

1H-T-NMR methods in conjunction with normally membrane-impermeable Mn2+ were used to study the effect of reductive methylation of specific lysine residues of Band 3, the major transmembrane protein, on water permeability. At 21 degrees C, the water apparent transverse relaxation time (T2) was decreased by nearly 16% (p less than .00001) for cells with modified Band 3. Atomic absorption measurements of control and methylated cells showed an increased level of Mn2+ in the erythrocyte cytosol following methylation. This increased level of this paramagnetic relaxation agent is sufficient to relax interior water protein to the values obtained. Thus, following specific methylation of band 3, increased membrane permeability to divalent cations is observed. The results are discussed with reference to possible conformation changes of Band 3 following methylation, and the findings are interpreted be mean that the conformation of Band 3 has influence on cation permeability to erythrocyte membranes.

Membrane processes associated with the osmotic-pulse incorporation of inositol hexaphosphate.

In previous studies (Biochem. Biophys. Res. Commun. 144, 779-786 (1987); Prog. Clin. Biol. Res. 292, 65-75 (1989)), we showed that inositol hexaphosphate (IHP), when added to erythrocyte membrane ghosts in the range 0.6-2.5 mM, caused a large disruption of skeletal protein-protein interactions as monitored by electron paramagnetic resonance techniques. IHP incorporated into intact cells by an osmotic-pulse method (J. Cell. Physiol. 129, 221-229 (1986)) leads to cells with markedly decreased oxygen affinity. Exposure of the red cells to higher levels of IHP during the osmotic pulse leads to less lysis and more normal cellular indices after healing of the transiently-disrupted membrane (J. Lab. Clin. Med. 113, 58-66 (1989)). In order to determine what effect higher levels of IHP had on skeletal proteins and bilayer lipids of membrane ghosts, spin labeling studies were performed. The main findings were: (a) There was a concentration-dependent alteration in skeletal protein interactions. At concentrations greater than 25 mM IHP, the effectiveness of IHP to disrupt skeletal protein interactions was diminished. (b) No apparent alteration of the motion or order of phospholipids or the lipid water interface of intact cells into which IHP was incorporated occurred, suggesting that higher levels of IHP do not alter the physical state of the lipid bilayer.