Membrane protein phosphorylation during stomatocyte-echinocyte transformation of human erythrocytes.

The normal, discoid shape of red blood cells represents an equilibrium between two opposing factors, i.e., stomatocytic and echinocytic transformations. Most stomatocytic agents were found to be inhibitors of calmodulin, a regulator of the phosphorylation of membrane proteins. We determined whether red cell shape transformations could be caused by changes in phosphorylation of membrane proteins, specifically the cAMP-dependent phosphorylation of ankyrin and band 4.1. Red blood cells were incubated with 32P and 100 microM chlorpromazine (stomatocytic transformation) or 30 mM sodium salicylate (echinocytic transformation) for various time intervals. Ghost membrane proteins were examined by polyacrylamide gel electrophoresis and autoradiography. Spectrin (beta-chain), ankyrin, band 3, band 4.1 and 4.9 were phosphorylated. No change was found in the degree and pattern of phosphorylation after stomatocytic transformation. Salicylate caused a reversible inhibition of transmembranous phosphate transport in both directions. The results indicate that the stomatocytic transformation induced by chlorpromazine and the echinocytic transformation induced by salicylate do not involve a change in phosphorylation, but that the echinocytic transformation induced by salicylate is associated with an inhibition of transmembranous transport of phosphate. Studies with salicylate suggest that the phosphorylation sites of band 3 are found mainly on the endofacial side of the membrane.

Regional cerebral metabolic activity in the rat following experimental subarachnoid hemorrhage.

A new experimental model was employed to investigate alterations of cerebral metabolic activity in rats subjected to extensive subarachnoid hemorrhage (SAH). The hemorrhages were produced in anesthetized animals by inserting 0.37 ml fresh autologous arterial blood into the subarachnoid space. Rats that underwent sham operations received subarachnoid injections of mock CSF to study the effects of sudden raised intracranial pressure (ICP). Forty-eight hours after subarachnoid injection, the unanesthetized rats were given intravenous injections of [14C]2-deoxyglucose. Experiments were terminated 45 min later by decapitation, and the brains were removed and frozen. Regional brain metabolic activity was studied employing quantitative autoradiography. In comparison with control animals, cerebral metabolic activity was diffusely decreased following SAH. Statistically significant decreases in metabolic activity of less than 34% were observed in 17 of 30 brain regions studied. The largest percentage reductions were in regions displaying the highest basal metabolic rates. Subarachnoid injections of mock CSF also produced depression of cerebral metabolic activity, but quantitatively these changes were not as pronounced as in the hemorrhage group. These studies demonstrate regional changes in brain function following SAH. The data relate these changes to both the presence of blood in the subarachnoid space and sudden raised ICP.

Time-dependent effects of endotoxin on the ultrastructure of aortic endothelium.

The aortic endothelium from control and Escherichia coli (E. coli) endotoxin-treated rats and rabbits was examined by transmission and scanning electron microscopy. Following the intravenous injection of endotoxin, the animals were sacrificed at intervals ranging from 1 min to 4 hr. As early as 1 min after endotoxin, there was a widening of the subendothelial space (SES) and an increase in tortuosity of the internal elastic lamina (IEL). At 5 min, the tortuosity of the IEL increased to a peak value, and the SES showed an increase in the amount of smooth muscle cells (SMC). Initial endothelial damage occurred 5 min after endotoxin: SEM showed some spindle-shaped endothelial cells starting to peel from the underlying SES, and TEM showed some endothelial cells protruding or arching into the lumen. The new findings in this study are that endotoxin injection a) has a very rapid (less than 15 min) effect on rat and rabbit aortic endothelium, including localized endothelial injuries in the intima, and b) induces ultrastructural alterations also in the SES, IEL and portions of the tunica media. These effects were largely reversed within 1 hr after endotoxin administration, thus indicating that the endothelium and other components of the arterial wall can recover with great speeds.

Effects of pressure on vesicle size, density, and distribution in the canine carotid arterial endothelium.

The purpose of the present experiment was to study the effects of internal hydrostatic pressure on vesicle size, density, and distribution in the canine carotid arterial endothelium by transmission electron microscopy. The pressures applied in this study were 0 (control), 40, 60, 80, 100, and 150 mm Hg. The results of transmission electron microscopy and computer analysis on the plasmalemmal vesicles of aortic endothelium showed that luminal, abluminal, and junctional vesicles all increased their diameter as the pressure was raised from 0 mm Hg, reaching a maximum at 80 mm Hg, and then decreased in size with further increases in pressure to 150 mm Hg. There was a significant difference in diameter among vesicles in different regions of the endothelium, with the diameter of luminal vesicles larger than those of abluminal and junctional vesicles. The densities of vesicles showed very little change from 0 to 80 mm Hg; but they increased markedly as the pressure was further raised from 80 to 150 mm Hg. These results indicate that pressure is an important mechanical factor governing the size and density of plasmalemmal vesicles in aortic endothelium.

Effects of sphere size and injection site on regional cerebral blood flow measurements.

Regional cerebral blood flows and shunting of microspheres with four different sizes (9,12, 16 and 25 microns) into the superior sagittal sinus were determined in twelve dogs. Venous blood was collected from the superior sagittal sinus for 120 min after the injection of microspheres, and the dogs were then sacrificed immediately. Results on blood flow measurements and venous shunting determinations were similar between left ventricular and left atrial injections. Blood flows measured by 12, 16 and 25 microns spheres were comparable in various brain tissues, except the choroid plexus. 9 microns spheres underestimated blood flows in all regions studied: by 13-19 percent in the cerebral cortex, midbrain, brain stem and cerebellum, by 34-42 percent in the cortical white matter, corpus callosum and cervical cord, and by 64-81 percent in pituitary gland and choroid plexus. These results probably reflect regional difference in microvascular architecture. Venous shunting of 9, 12, 16 and 25 microns spheres during a 24 hr period were 23.6 +/- 2.5, 12.6 +/- 1.2, 4.8 +/- 1.4, and 4.0 +/- 1.2 percent (mean +/- SEM), respectively, with respect to the arterial delivery. Although most of the venous shunting occurred during the first 3 min after the injection of microspheres, it continued 3-60 min after the injection. Beyond 60 min, the venous shunting became minimal for 16 and 25 microns spheres, while significant amount of 9 microns spheres continued to appear in sagittal sinus. This time dependent shunting indicates that some microspheres may be transiently trapped in the microcirculation and become gradually dislodged with time. Failure to consider this time dependence may underestimate the shunting of microspheres through the microcirculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Determinations of blood flow and shunting of 9- and 15-micrometer spheres in regional beds.

In 17 pentobarbitalized dogs, the shunting of 15-micrometer and 9-micrometer microspheres was studied in the brain, myocardium, kidney, intestine, and lung. The veins of these organs were catheterized for constant blood withdrawal for 2 min by direct venipuncture. The ratio of microsphere radioactivity in the venous blood to that in the arterial blood gave the shunting of microspheres by the venous sampling technique. The 15-micrometer microspheres showed 2% or less shunting for all organs studied, whereas the 9-micrometer microspheres had shunting ranging from 3% in the coronary sinus to 24% in the portal vein. The shunting of 9-micrometer microspheres was also calculated from direct tissue counting, where the 15-micrometer spheres were considered to be completely entrapped. The results of direct tissue counting indicate that the 2-min venous sampling underestimates microsphere shunting. CO2 administration increased significantly the shunting of 9-micrometer spheres, whereas the shunting of 15-micrometer spheres determined by venous sampling remained less than 2%. Consideration of shunting indicates that the 15-micrometer microspheres might be more appropriate for regional organ blood flow measurements, including the myocardium.

Baroreflex control of heart rate in humans during nitroprusside-induced hypotension.

The baroreflex control of heart rate was investigated on 10 informed human subjects during light halothane anesthesia (0.3-0.5%, inspired concentration). The relationship of systolic pressure (SP) to the succeeding pulse interval (PI) was evaluated on a beat-to-beat basis during the entire course of sodium nitroprusside (SNP) depressor test. The initial slope of SP-PI plot (dPI/dSP) was used as an index of the sensitivity of baroreflex control of heart rate. Following an injection of SNP (4-6 micrograms/kg), dPI/dt was related directly to dPI/dSP, whereas the latter was inversely correlated with dSP/dt. The recovery of PI lagged behind that of SP, and there was a hysteresislike loop on the SP-PI plot. The time lag of PI recovery and the loop of SP-PI plot were markedly decreased by propranolol treatment and significantly increased by atropine. The slopes of SP-PI plot were significantly decreased by atropine but relatively unaffected by propranolol. These results indicate that SNP-induced hypotension in man during halothane anesthesia is associated with a withdrawal of parasympathetic inhibition and an enhancement of sympathetic activity. The autonomic control of heart rate in response to rapid changes in arterial pressure induced by SNP is dominated by parasympathetic influence; the more persistent sympathetic activity only becomes evident when the parasympathetic influence subsides quickly as the arterial pressure stays relatively constant at a new level. The slope of SP-PI plot (dPI/dSP) and the ratio of dPI/dt to dSP/dt during the decreasing pressure phase of SNP test can be used as indices for the sensitivity of baroreflex control of heart rate.

Effects of hematocrit variations on regional hemodynamics and oxygen transport in the dog.

The responses of alterations in regional hemodynamics and oxygen transport rate to hematocrit (Hct) were studied in 20 pentobarbitalized dogs. Hemodilution was carried out by isovolemic exchange with plasma in 12 dogs and the hemoconcentration with packed cells in 8 dogs. The cardiac output and regional blood flows were determined with the microsphere technique. In hemodilution, the increases of blood flow to the myocardium and the brain were out of proportion to the increase of cardiac output; the oxygen supply to the myocardium remained unchanged while that to the brain decreased only slightly. In hemoconcentration, vasodilation occurred in the myocardium and the brain to maintain constant oxygen supply. Splenic vessels had marked vasoconstriction with Hct alteration in either direction. Blood vessels in the liver, intestine, and kidney responded with a milder vasoconstriction and maintained a constant oxygen supply between Hct of 30-55%. Therefore, during Hct alteration, redistribution of blood flow to myocardium and brain occurred. The optimal Hct range for constant oxygen supply was different among various organs.

Regional cerebral blood flow and oxygen consumption of the canine brain during hemorrhagic hypotension.

The sequential changes in systemic and cerebral hemodynamics, systemic and cerebral oxygen transport and consumption rates, and the regional blood flows (measured with 15 micron microspheres) to the cortical and subcortical brain tissues were determined in nine dogs subjected to graded hemorrhage (10 ml/kg X 4 at 15 min intervals). As hemorrhage progressed, both mean arterial pressure and cardiac output decreased progressively. In contrast to the systemic circulation, the mean cerebral blood flow (mCBF) was well maintained by cerebral vasodilation and the cerebral O2 consumption rate (CMRO2) increased during the first three stages of hemorrhage. At 40 ml/kg of hemorrhage, there were significant reduction in mCBF and CMRO2 despite the increase in O2 extraction, suggesting the occurrence of cerebral hypoxia and decompensation of the cerebral circulation. There were remarkable regional variations in the responses of regional cerebral blood flows (rCBF) to hypovolemia, resulting in a significant redistribution of cerebral blood flow. The fractions of cardiac output supplying the diencephalon (thalamus and hypothalamus), the brain stem (pons and medulla oblongata) and the cervical spinal cord increased after hemorrhage up to 40 ml/kg. The redistribution of rCBF favors those areas where neurons related to cardiovascular control are located. These findings have significant implications relating to hemodynamic regulation during hemorrhagic hypotension.

Influence of physicochemical factors on rheology of human neutrophils.

The effects of variations in temperature, pH, and osmolality on the rheological properties of human neutrophils were determined by studying the cell deformation in response to aspirational pressure applied via a micropipette. The time history of the deformation was analyzed by the use of a standard solid viscoelastic model consisting of an elastic element K1 in parallel with a Maxwell element (an elastic element K2 in series with a viscous element mu). With changes in temperature over a range of 9-40 degrees C, only mu varied inversely with temperature, while K1 and K2 did not show significant alterations. Variations in pH over the range of 5.4-7.8 did not significantly affect the viscoelastic coefficients, but K1 and mu rose at pH 8.4. An increase in osmolality caused all three coefficients to rise, but a decrease in osmolality had relatively little effect on the coefficients. These changes in response to physicochemical variations serve to provide insights into the viscoelastic properties of neutrophils and their possible roles in health and disease.