Biomedical subjects
Publications and source records attributed to T M McIntyre.
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The effect of cessation of net phospholipid synthesis on the cytoplasmic and outer membranes of Escherichia coli was investigated in a mutant strain defective in the first enzyme of phospholipid synthesis, the sn-glycerol-3-phosphate (glycerol-P) acyltransferase. The glycerol-P (glycerol) auxotropic phenotype of this strain resulted from an altered membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of the parental activity. When net phospholipid synthesis was halted during glycerol deprivation, both soluble and cell envelope protein synthesis continued. Fractionation of the membranes derived from glycerol-supplemented and glycerol-deprived cultures by isopycnic banding in sucrose gradients revealed that both the cytoplasmic and outer membranes of the deprived culture banded at higher buoyant densities. The protein/phospholipid ratio of both the cytoplasmic and outer membranes increased approximately 60% during the period of glycerol deprivation. The distribution of two cytoplasmic membrane activities, NADH oxidase and 1-acylglycerol-P acyltransferase, and an outer membrane activity, phospholipase A1, showed that the total membranes derived from glycerol-deprived cultures were separated cleanly into cytoplasmic and outer membrane fractions. Both cytoplasmic and outer membrane proteins were synthesized and integrated into their respective membranous structures when net phospholipid synthesis was halted. Hence, the biosynthesis of membrane phospholipid and membrane protein are not tightly coupled. Further, these data suggest that cellular control mechanisms exist which maintain the protein content of both membranous structures below the point where they are saturated with protein.
Atherosclerosis has an underlying inflammatory component. Oxidation of low-density lipoprotein (LDL) particles to modified forms promotes atherogenesis by supplying cholesterol and through the oxidative generation of agents that activate macrophages, smooth muscle and endothelial cells. A primary target of oxidizing compounds, derived from cigarette smoke, dietary sources, exuberant inflammatory cell responses and normal cellular metabolism among other sources, are the esterified polyunsaturated fatty acids in the phospholipid shell that surrounds the insoluble lipids of the lipoprotein core. One type of phospholipid oxidation product mimics the structure of the potent inflammatory mediator platelet-activating factor (PAF), and these oxidation products activate the PAF receptor found on platelets, monocytes and leukocytes. Production of such PAF mimetics is, in contrast to the physiologic generation of PAF, uncontrolled. PAF mimetics and other phospholipid oxidation products are found in atherosclerotic lesions or even in blood after exposure to cigarette smoke. Here we summarize our data describing the structure, activity and metabolism of the PAF-like lipids found in atherogenic LDL particles.
Permeabilization is an important tool in cell biology that allows manipulation of intracellular mechanisms by introduction of probes and regulatory molecules into the cell cytoplasm. We found that incubation of endothelial cells (ECs) with glass beads resulted in nonspecific permeabilization of human and bovine ECs without removal of the cells from monolayer culture. This poration of the plasma membrane allowed the introduction of macromolecules (dextrans less than or equal to 152 kd and immunoglobulins) as well as small, charged molecules (Lucifer Yellow). We found that nonspecific permeabilization of the EC was transient and defined the conditions under which integrity of the plasma membrane was reestablished. This process was dependent on time, temperature, and the presence of extracellular calcium. We also demonstrated that permeabilized ECs regain functional characteristics. This was defined by a number of criteria, including the ability to rapidly reestablish confluent monolayer morphology, to extrude Lucifer Yellow, to adhere and spread after passage, and to synthesize biologically active molecules after stimulation with a receptor-mediated agonist. Thus, transiently porated ECs loaded with appropriate probes can be used in studies of regulatory mechanisms while remaining in monolayer culture, a condition in which many phenotypic features are similar to in situ endothelium. Furthermore, the porated EC may be a useful model for defining the mechanisms that influence the repair of endothelial plasma membrane injury.
Stimulation of cultured bovine endothelial cells from aorta, pulmonary artery, coronary artery, and vena cava with calcium ionophore A23187, adenosine triphosphate (ATP), bradykinin, and angiotensin II resulted in production of platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. In pulmonary artery endothelial cells, maximal accumulation occurred within 20 minutes, and the PAF remained cell-associated. This response was concentration-dependent with a maximal effect at 10(-8)M bradykinin and 10(-6)M angiotensin II. Stimulation of in situ endothelial cells on isolated strips of bovine pulmonary artery with calcium ionophore, ATP, and bradykinin also resulted in production of PAF. In cultured pulmonary artery endothelial cells, PAF appeared only in response to the same agonists that caused prostacyclin (PGI2) release. These findings are similar to earlier studies with human umbilical vein endothelial cells and suggest that the production of PAF and the arachidonate metabolite PGI2 are tightly linked in these vessels. Aortic endothelial cells, both in culture and on isolated strips of aorta, also produced PAF in response to bradykinin in a time-and concentration-dependent fashion. Cultured endothelial cells from coronary vessels and the inferior vena cava accumulated PAF when appropriately stimulated as well. The finding that both cultured endothelial cells and in situ, ex vivo endothelium from diverse vascular sites produce PAF when appropriately stimulated suggests that this is a generalized response of endothelial cells. Because PAF has potent vasoactive, pro-aggregatory, and pro-inflammatory effects, the results from these experiments suggest that the local accumulation of this lipid may be important in vascular injury and inflammation.