The effect of a combination of medetomidine-butorphanol and medetomidine, butorphanol, atropine on glomerular filtration rate in dogs.

The effects of intramuscularly administered medetomidine and butorphanol (MB), and medetomidine, butorphanol, atropine (MBA) on glomerular filtration rate (GFR) were determined in six dogs as measured by 99m-Tc-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA) nuclear scintigraphy. Direct systolic, diastolic, and mean arterial blood pressures and heart rate were measured at regular time intervals before, during, and after GFR calculations. The mean GFR measurement following MB was significantly greater (4.44 ml/min/kg) than following MBA (3.82 ml/min/kg) or saline treatment (3.41 ml/min/kg). There was no significant difference between the mean GFR measurements following MBA injection and following saline injection. Diastolic and mean arterial pressures following MBA injection were significantly higher than the values recorded after either MB or saline alone. Heart rate following MB administration was significantly lower than that recorded for dogs receiving MBA or saline alone. The results of this study indicate that the administration of medetomidine in combination with butorphanol significantly increases total GFR in healthy dogs, while the administration of the combination of medetomidine, butorphanol, and atropine does not.

Protein phosphatase 2B inhibitor potentiates endothelial PKC activity and barrier dysfunction.

Serine/threonine (Ser/Thr) protein phosphatases (PPs) are implicated in the recovery from endothelial barrier dysfunction caused by inflammatory mediators. We hypothesized that Ser/Thr PPs may regulate protein kinase C (PKC), a critical signaling molecule in barrier dysfunction, in the promotion of barrier recovery. Western analysis indicated that bovine pulmonary microvascular endothelial cells (BPMECs) expressed the three major Ser/Thr PPs, PP1, PP2A, and PP2B. Pretreatment with 100 ng/ml of FK506 (a PP2B inhibitor) but not with the PP1 and PP2A inhibitors calyculin A or okadaic acid potentiated the thrombin-induced increase in PKC phosphotransferase activity. FK506 also potentiated thrombin-induced PKC-alpha but not PKC-beta phosphorylation. FK506 but not calyculin A or okadaic acid inhibited recovery from the thrombin-induced decrease in transendothelial resistance. Neither FK506 nor okadaic acid altered the thrombin-induced resistance decrease, whereas calyculin A potentiated the decrease. Downregulation of PKC with phorbol 12-myristate 13-acetate rescued the FK506-mediated inhibition of recovery, which was consistent with the finding that the thrombin-induced phosphorylation of PKC-alpha was reduced during the recovery phase. These results indicated that PP2B may play a physiologically important role in returning endothelial barrier dysfunction to normal through the regulation of PKC.

Aminothiol WR-1065 protects endothelial cell morphology against alterations induced by lipopolysaccharide.

In septic patients, lipopolysaccharide (LPS) damages the vascular endothelium, which manifests as tissue edema and impaired healing. This pathology occurs when LPS distorts endothelial cell morphology partly by generating free radicals. A radioprotector that scavenges free radicals, the aminothiol WR-1065 ([N-2-mercaptoethyl]-1-3-diaminopropane) was found in a prior study to normalize the morphology of irradiated endothelial cells (Mooteri SN, Podolski JL, Drab EA, et al: Radiat Res 145:217-224, 1996). The aim of this study was to determine whether WR-1065 also normalized endothelial cell morphology following exposure to LPS. For this aim, portions of bovine aortic endothelial cell cultures were denuded and exposed to LPS at 1 ng/mL. After 30 min, the apical membrane expressed increased integrin receptor to fibronectin, alpha5beta1. After 5 h, the morphology of the cells at the leading edge was distorted, and cell-cell contact was lessened. Also, filamentous actin-containing stress fibers were dissipated; however, filamentous actin content per cell was unchanged. Treatment with 2 mM WR-1065 for 2 h prior to LPS exposure attenuated the increased expression of alpha5beta1 and promoted cell-cell contact in the migrating endothelial cells. WR-1065 also promoted the retention of stress fibers and actin cytoskeletal shape in cells treated with LPS. Thus, LPS distorted endothelial cell morphology after increasing apical membrane expression of alpha5beta1 and dissipating stress fibers, effects prevented by WR-1065.

Recent advances in peptic ulcer disease: Helicobacter pylori infection and its treatment.

The identification of the gastric bacterium Helicobacter pylori was a significant advancement in the treatment of peptic ulcer disease. H. pylori infects the gastric mucosa and its eradication is associated with the prevention of ulcer recurrence. The significance of this finding is that bacterial infections can be treated and cured, offering hope to individuals with peptic ulcers. Characteristics of H. pylori and its putative role in ulcer formation are discussed. The recent challenge has been to identify a drug regimen that will effectively eradicate this organism inexpensively and conveniently, while not causing significant side effects. Current diagnostic methods and antimicrobial therapies for H. pylori are also presented. A look to future directions in therapies includes a hoped-for vaccine to prevent gastric infections with H. pylori.

Remodeling the shape of the skeleton in the intact red cell.

The role of the membrane skeleton in determining the shape of the human red cell was probed by weakening it in situ with urea, a membrane-permeable perturbant of spectrin. Urea by itself did not alter the biconcave disk shape of the red cell; however, above threshold conditions (1.5 M, 37 degrees C, 10 min), it caused an 18% reduction in the membrane elastic shear modulus. It also potentiated the spiculation of cells by lysophosphatidylcholine. These findings suggest that the contour of the resting cell is not normally dependent on the elasticity of or tension in the membrane skeleton. Rather, the elasticity of the skeleton stabilizes membranes against deformation. Urea treatment also caused the projections induced both by micropipette aspiration and by lysophosphatidylcholine to become irreversible. Furthermore, urea converted the axisymmetric conical spicules induced by lysophosphatidylcholine into irregular, curved and knobby spicules; i.e., echinocytosis became acanthocytosis. Unlike controls, the ghosts and membrane skeletons obtained from urea-generated acanthocytes were imprinted with spicules. These data suggest that perturbing interprotein associations with urea in situ allowed the skeleton to evolve plastically to accommodate the contours imposed upon it by the overlying membrane.

WR-1065 and radioprotection of vascular endothelial cells. II. Morphology.

Although the aminothiol WR-1065 protects normal tissues, its direct effect on the damage and restoration of the vascular endothelium is not clear. In endothelial cells, WR-1065 attenuates both the DNA damage and the G1-phase arrest induced by radiation. After the destruction of nearby endothelial cells, the survivors rearrange their cytoskeleton, migrate and replicate. To determine the effect of radiation on morphology and migration, portions of bovine aortic endothelial cell cultures were denuded with a pipette tip and irradiated (137Cs gamma rays). The following observations were noted after 5 Gy: within 10 min, there was increased formation of protein-mixed disulfides including actin-mixed disulfide; after 30-min, alpha 5 beta 1, the integrin receptor for fibronectin, was up-regulated on the apical membrane surface. Within 5 h, actin-containing stress fibers reorganized, although there was no change in the total filamentous (F-)actin content within the cells. Compared to controls after 24 h, the irradiated cells had migrated 15% farther (P < 0.01), and at the leading edge covered twice the surface area (P < 0.0001). The addition of 2 mM WR-1065 for 2 h before 5 Gy inhibited the increased expression of alpha 5 beta 1, promoted retention of stress fibers and prevented the enhanced cell migration and spreading. These results indicate that WR-1065 prevents radiation-induced morphological responses. This effect appears to be mediated by an impact on both adhesion molecule expression and cytoskeletal reorganization.

Length distribution of F-actin in Dictyostelium discoideum.

Inhibition of deoxyribonuclease I activity was used to assay the actin monomers and the pointed ends of actin filaments in lysates of Dictyostelium discoideum. The KD for the binding reaction was 0.2-0.3 nM. Total cellular actin was 93 microM in monomers (approximately 0.1 fmol/cell) of which roughly half was initially polymeric. Essentially all of the filamentous actin (F-actin) was readily pelleted in the microcentrifuge and was therefore presumed to be in the cytoskeleton. Free F-actin barbed ends, measured as pelletable [3H]cytochalasin B, numbered 1.8 x 10(5)/cell; nuclei for the polymerization of rabbit muscle globular (monomeric) actin numbered 2.0 x 10(5)/cell; and pointed ends, determined by their inhibition of deoxyribonuclease I, numbered 3.6 x 10(5)/cell. These values suggest that half the barbed ends might be occluded. On average, the filaments contained approximately 76 subunits and were therefore about 0.2 micron long. The distribution of their lengths was estimated from the time course of depolymerization following vast dilution. Three populations were defined. In one experiment, the smallest population contained 71% of the F-actin mass and 96% of the pointed ends; these filaments averaged 80 subunits or 0.22 microns in length. An intermediate population contained 14% of the F-actin mass and 3% of the filaments; these were roughly 460 subunits (1.3 microns) long. The largest population contained 15% of the F-actin mass in about 0.3% of the filaments; these were 13 microns in length, about the diameter of the cell. The numerous short filaments might populate a cortical mesh, while the long filaments might constitute endoplasmic bundles.

Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons.

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.

Erythrocyte membrane lysophospholipase activity in muscular dystrophy.

The membrane lysophospholipase activity of erythrocytes obtained from Duchenne muscular dystrophy patients was lower than that of erythrocytes from age-matched normal boys. On the other hand, the membrane enzyme activity of erythrocytes from myotonic dystrophy patients was not different from that of their age- and sex-matched controls. Dipyridamole (0.1 mM) and glycerol 3-phosphorylcholine (2 mM) had no significant effect on these enzyme activities. These results suggest that membrane lysophospholipid metabolism may be altered in Duchenne muscular dystrophy but not in myotonic dystrophy.

Lysophosphatidylcholine-induced lysis of erythrocytes in Duchenne and myotonic dystrophies and in Huntington's disease.

Erythrocytes from Duchenne dystrophy patients lysed more readily than red cells from age-matched normal boys when lysophosphatidylcholine (LPC) concentrations that caused 50% lysis were compared. Erythrocytes from myotonic dystrophy patients appeared to be more resistant than cells from age-matched normal adults at certain medium LPC concentrations. Erythrocytes from patients with Huntington's disease showed no significant differences from erythrocytes of normal adults. Thus, the manner in which erythrocytes respond to LPC may reflect the putative membrane alterations in these diseases. Inhibition of LPC-induced lysis by 0.1 mM dipyridamole was observed in all groups. Since this agent did not inhibit LPC lysis at 0 degrees C, its action at 37 degrees C could be related to activation of a membrane enzyme. On the other hand, dipyridamole decreased osmotic fragility at 0 degrees C and 37 degrees C indicating that a physical change in membrane structure may be the primary alteration produced by this agent.