Production of 5-hydroxyindoleacetic acid from serotonin by cultured endothelial cells

Endothelial cells cells from bovine aorta and human umbilical vein and fibroblasts from human foreskin were cultured and subsequently evaluated for ability to metabolize serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA). Cells were incubated for three hours with 4 X 10(-6) M [14C] 5-HT creatinine sulfate. [14C] 5-HIAA was separated from labeled 5-HT by column chromatography and measured for scintillation counting. Production of 5-HIAA by bovine aorta cells was 39.0+/-7.5 (S.E.M., n=6) nmoles per 10(9) cells per hour. Production of 5-HIAA was markedly inhibited by the presence of 10(-4) M iproniazid (an inhibitor of monoamine oxidase) or 10(-4) M imipramine (an inhibitor of amine transport). 5-HIAA was the only product of 5-HT metabolism detected by thin layer chromatography. Production of 5-HIAA by human umbilical vein endothelial cells was 5.4+/-2.0 nmoles per 10(9) cells per hour (n=5) and by human foreskin fibroblasts was 3.9+/-1.4 nmoles per 10(9) cells per hour (n=5). The results obtained during incubation in the presence and absence of inhibitors indicate that bovine aorta endothelial cells maintained in tissue culture are able to transport serotonin with subsequent production of 5-HIAA. By contrast, human umbilical vein endothelial cells and fibroblasts exhibited relatively low rates of 5-HT uptake and metabolism.     

Contraction of vascular smooth muscle in cell culture

The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells. This limitation has been overcome by the cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane). Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist-induced contraction. Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2-dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture.     

Production and characterization of a monoclonal antibody to human type III collagen

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.     

Smooth muscle trans-membrane sarcoglycan complex in partial bladder outlet obstruction

The urinary bladder experiences both distension and contraction as a part of the normal filling and emptying cycle. To empty properly, tension generated intracellularly in a smooth muscle cell must be smoothly and efficiently transferred across its sarcolemma to the basement membrane, which mediates its binding to both the extracellular matrix and to other cells. As a consequence of urethral obstruction, the bladder cannot generate appropriate force to contract the organ, thereby leading to inefficient emptying and associated sequelae. In this study, an animal model of urethral obstruction was utilized to study the membrane-associated structures that transfer tension across the sarcolemma of bladder smooth muscle cells. Immunohistochemical localization of key components of the smooth muscle tension transfer apparatus (TTA) was performed utilizing specific antibodies against:(1) the α-chains of type IV collagen, a basement membrane component, and (2) β-sarcoglycan, an integral membrane protein that is a participant in the physical linkage between the cytoskeleton and the basement membrane. We demonstrate, in obstructed animals, that there is a pronounced disruption of the TTA with a physical displacement of these two components that can be demonstrated at the level of the light microscope using scanning confocal microscopy. Electron microscopy further demonstrates significant increases in the size of the junctional plaques between smooth muscle cells.     

Characterization of a fibroblast cell from the urinary bladder wall

Summary For the first time we report on the growth, culture, and matrix production characteristics of a cell type isolated from the lamina propria of the urinary bladder wall. A fibroblastlike cell was identified as distinct from bladder detrusor smooth muscle cells and urothelium based on morphology, growth characteristics, and immunohistochemical staining. Characterization of extracellular matrix synthesis by this cell type using³⁵S-methionine metabolic labeling demonstrated that these cells are capable of secreting components of the surrounding connective tissue, including several fibrillar collagens, a basement membrane collagen, and fibronectin.     

A system to reproduce and quantify the biomechanical environment of the cell

An in vitro system that permits application of a uniform biomechanical stimulus to a population of cells with great precision has been developed. The device is designed to subject living cells to reproducible and quantifiable biaxial strains from 0 to 10% at rates from quasi-static to 1 s-1 and frequencies from 0 to 5 Hz. Equations for determining the strain in the substrate upon which the cells are grown, based on easily measured parameters, are derived and validated experimentally. The mechanical properties of the substrate are determined, and it is demonstrated that cells can easily be cultured in the apparatus. By use of the system, cloned bovine pulmonary artery endothelial cell clones are subjected to 5% biaxial strains applied at a peak strain rate of 0.5 s-1 and a frequency of 1 Hz for 7 h with cell viability greater than 84% and cell detachment less than 8%. We demonstrate that cells must be attached to the substrate for them to be stretched and that cell strain and substrate strain are not equal. With the use of fluorescently labeled beads as cell surface markers to measure the actual strain produced in the cells as a result of the deformation of the substrate, cell elongation was found to be approximately 60% of the strain in the substrate. This constant appeared to be affected by both in vitro cell age and morphology.     

Collagen synthesis by cloned pulmonary artery endothelial cells

Pulmonary artery endothelial cells were isolated from bovine fetal blood vessels and used for biosynthetic studies. At confluence, cultures were incubated in minimal essential medium (MEM) without serum containing [U-14C]proline. After 24 hours, medium was removed and labeled proteins were precipitated by the addition of ammonium sulfate and fractionated by diethylaminoethyl (DEAE)-cellulose chromatography. The elution profile showed four major peaks and one minor peak. Fractions within each peak were pooled, subjected to digestion by chymotrypsin and/or collagenase, and analyzed by polyacrylamide gel electrophoresis. Peak l contained a collagen which contained approximately 6% of the 3-hydroxyproline isomer while total hydroxyproline content was approximately 45%. This material was digested by purified bacterial collagenase and had a mobility slightly slower than that of alpha 1(III) which did not change under conditions that reduce disulfide bonds. Upon digestion with chymotrypsin under conditions where native procollagens are converted to alpha-chains, this material was digested. These properties suggest that this material is type VIII or EC (endothelial cell) collagen. Peak 2 contained substantial fibronectin while peak 3 contained primarily type III procollagen. The last major peak contained a mixture of collagenous and noncollagenous material. Upon digestion with chymotrypsin, several peptides were generated which were sensitive to bacterial collagenases. The two major chymotrypsin-resistant components had mobilities slower than that of alpha(III) and were not disulfide-bonded.