Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line.

The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph. Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth. The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL). U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL. This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor. The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis. Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow. Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis. However, if the cells are incubated in the presence of no less than 40 micrograms LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated. The effect of LDL is specific since neither VLDL nor HDL3 at the same protein concentration activates horseradish peroxidase uptake by the cells. Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects. The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish peroxidase binding and uptake by adsorptive endocytosis. The rate of fluid endocytosis and endosome formation seems to depend on cellular cholesterol content presumably because cholesterol is involved in maintaining the appropriate plasma membrane structure and fluidity.

Acute effect of substance P in immunologic vasculitis in the rat colon.

Substance P has been implicated as a neuronal mediator of inflammation in various inflammatory conditions. However, the exact role played by substance P in inflammatory bowel diseases or in experimental colonic vasculitis has not been clearly understood. In this study, we examined the effect of close superior mesenteric artery injection of substance P under prevailing inflammatory conditions induced by intravenous human albumin antialbumin immune complex followed by intracolonic perfusion of 2.5% formaldehyde in rats or intracolonic perfusion of 5% alcohol alone. The immune complex- and formaldehyde-treated rats showed severe microvascular changes such as microvascular plugging by red blood cells, endothelial breakage and extravasation of plasma proteins and red blood cells. The bolus injection of 10(-8) M substance P reduced extravasation of Evans blue dye by 50% and the tissue wet to dry ratio by 20% in immune complex- and formaldehyde-perfused rats. Myeloperoxidase activity was not changed. Substance P also significantly inhibited (44%) the extravasation in alcohol-perfused rats. Pretreatment of immune complex- and formaldehyde-treated rats with substance P antagonist reversed the effect of substance P. These findings suggest that the most immediate effect of substance P may be vasodilation and clearing of vascular plugs induced by immune complex and formaldehyde. This effect of substance P differs from its chronic effect, which causes vasodilation and extravasation.

Morphologic study of the blood vessels of the superior cervical ganglion of the albino rat.

Blood vessels of the rat superior cervical ganglion were examined by both light and electron microscopy. Direct blood supply to the superior cervical ganglion was derived from a capsular plexus of vessels. Intraganglionic vessels were for the most part capillaries. Some of these capillaries appeared dilated and sinusoidal. Although the ganglion did not seem to be densely vascularized, there was sufficient distribution to accommodate the nerve cell bodies of the ganglion. Individual capillaries served groups of neurons. Occasionally, capillary loops could be observed to surround single neuron perikarya. Ultrastructural studies revealed the presence of two types of capillaries. The majority of the capillaries of the rat superior cervical ganglion demonstrated a continuous, non-fenestrated endothelium. Typical junctional complexes were found on abutting endothelial surfaces. Endothelial flaps and microvilli were also observed on the luminal surface of some of the vessels. Numerous micropinocytotic vesicles were observed on both the luminal and abluminal surfaces of the endothelium. A small number of capillaries demonstrated a fenestrated endothelium. In both types of capillaries there was a basement membrane and an extracellular space containing collagen. Perikaryal cytoplasm was separated from the extracellular space by a thick layer of satellite cell cytoplasm.

Evidence for a blood-ganglion barrier in the superior cervical ganglion of the rat.

The permeability of the blood vessels in the superior cervical ganglion of the rat was tested by intravenous injection of horseradish peroxidase (HRP). By light microscopy, peroxidase activity was found in three locations: in the capsule of the ganglion, in the lumina of the blood vessels, and within macrophages. Electron microscopy revealed that virtually all ganglionic blood vessels contained HRP 5 minutes following its administration. The intensity of peroxidase activity declined over the period of 15 minutes. The enzyme was localized on the luminal surface of the endothelial cells, attaching to the glycocalyx. Endothelial microvilli, projecting into the vessel lumen, were also covered with peroxidase. Micropinocytotic vesicles on the luminal surface of the endothelium contained reaction product. Some of these vesicles were free within the cytoplasm of the endothelium but none was observed on the abluminal surface. Peroxidase activity was not detected in the extracellular space even after 15 minutes. The majority of blood vessels in the superior cervical ganglion possess a continuous endothelium with tight junctions; features associated with the blood-brain barrier of the central nervous system and peripheral nerves. It is proposed that these vessels perform a barrier function between the capillary circulation and the superior cervical ganglion.

Distribution of intravascularly injected lanthanum ions in ganglia of the autonomic nervous system of the rat.

Intravascular injection of lanthanum revealed that tight junctions of capillaries in sympathetic ganglia are impermeable to small ions and thus behave like capillaries of the blood-brain barrier. The failure of lanthanum to accumulate in the extracellular space suggests that fenestrated capillaries are not as ion-permeable as use of horseradish peroxidase (HRP) by some authors has indicated. A possible toxic action associated with high concentrations of HRP may be responsible for the high permeability of this substance. Testing with lanthanum demonstrated that sympathetic ganglia possess anatomic features that provide a hematic barrier. The blood-ganglion barrier resembles, but has not yet been demonstrated to be as absolute as the blood-brain barrier.

Electrostimulation and morphologic study of the nerves to the bone marrow of the albino rat.

The innervation of the bone marrow of the albino rat was investigated by electrostimulation and morphological methods. Stimulation of the lumbar sympathetic trunks resulted in the release of reticulocytes and neutrophils into the circulating blood. The effects of stimulation on other cell types in the bone marrow could not be definitely established. It was concluded that the nerve fibers to the bone marrow were distributed to the arteries. It is postulated that the transmitter substance released at the autonomic nerve endings may have an effect upon the permeability of the venous sinusiods and the mobility of the blood cells in the marrow parenchyma resulting in their release into the circulating blood.