This paper describes the localization of isomyosins in the pericytes of four rat microvascular beds: heart, diaphragm, pancreas, and the intestinal mucosa, by use of immunoperoxidase techniques and IgGs specific for either nonmuscle or smooth muscle isoforms. Based on the semiquantitative nature of the peroxidatic reaction, we concluded that the amount and distribution of these isoforms vary with the microvascular bed and also with vascular segments within the same bed. In the pericytes of small capillaries, nonmuscle isomyosin is the predominant form, whereas the smooth muscle isomyosin is present in very low concentration. A reversed relationship is found in the pericytes associated with larger capillaries and postcapillary venules. These results, taken together with previous findings on actin (Herman, I., and P. A. D'Amore, 1983, J. Cell Biol. 97:278a), tropomyosin (Joyce, N. C., M. F. Haire, and G. E. Palade, 1985, J. Cell Biol. 100:1379-1386, and cyclic GMP-dependent protein kinase (Joyce, N., P. DeCamilli, and J. Boyles, 1984, Microvasc. Res. 28:206-219), indicate that pericytes contain proteins essential for contraction in higher concentration than any other cells associated with the microvasculature, except smooth muscle cells. Pericytes appear to be, therefore, cells differentiated for a contractile function within the microvasculature.It has been repeatedly assumed that the pericytes of blood capillaries and postcapillary venules function as contractile elements within the microvasculature because of their morphologic and topographic characteristics (3,9,12,16,18,23,33). To test this assumption, the presence of significant amounts of essential contraction-associated proteins in pericytes must be established, and eventually their contraction and its effects on microvascular blood flow must be demonstrated directly in vivo.In our laboratory, two proteins associated with the regulation of contraction have been localized in microvascular pericytes by use of immunoperoxidase techniques. The companion paper (17) describes the localization of tropomyosin in the pericytes of four rat microvascular beds by use of an IgG specific for the smooth muscle isoform of this protein. In addition, we have localized cyclic GMP-dependent protein kinase, an enzyme postulated to function in the regulation of contraction in smooth muscle cells (14,19,27) pericytes by electron microscopic techniques (21, 32), and both smooth muscle and nonmuscle isoactins have been localized in pericytes in situ and in cell culture by immunofluorescence procedures (I 5).To complete the identification of essential contractile proteins in pericytes, it is necessary to determine whether they contain myosin. Available information on this topic is either indirect or insufficient. In rat brain pericytes, LeBeux and Willemot (2 l) have observed thick, tapering fibrillar structures with morphological and solubility characteristics similar to those of myosin filaments, and brief preliminary reports (8, 25) have been published on the immunocyto...
In these studies we have compared the relative amounts and isoforms of tropomyosin in capillary and postcapillary venule pericytes, endothelial cells, and vascular smooth muscle cells in four rat microvascular beds: heart, diaphragm, pancreas, and the intestinal mucosa. The results, obtained by in situ immunoperoxidase localization, indicate that (a) tropomyosin is present in capillary and postcapillary venule pericytes in relatively high concentration; (b) the tropomyosin content of pericytes appears to be somewhat lower than in vascular smooth muscle cells but higher than in endothelia and other vessel-associated cells; and (c) pericytes, unlike endothelia and other nonmuscle cells, contain detectable levels of tropomyosin immunologically related to the smooth muscle isoform. These results and our previous findings concerning the presence of a cyclic GMP-dependent protein kinase (Joyce, N., P. DeCamilli, and J. Boyles, 1984, Microvasc. Res. 28:206-219) in pericytes demonstrate that these cells contain significant amounts of at least two proteins important for contraction regulation. Taken together, the evidence suggests that pericytes are contractile elements related to vascular smooth muscle cells, possibly involved, as are the latter, in the regulation of blood flow through the microvasculature.Pericytes are polymorphic cells closely associated with the walls of capillaries and postcapillary venules. They have a cell body which is often highly elongated, and multiple branching foot processes which partially encircle the vessel wall. Pericytes can be distinguished from other perivascular cells, such as adventitial fibroblasts, by their location within the basement membrane of the vessels, and by the close apposition of the tips of their processes to the underlying endothelium. Electron microscopic studies have identified pericytes in a number of tissues and organs (I0, 12, 20, 36). They may, in fact, be common to all microvascular beds, although their relative frequency and distribution appear to vary from one microvascular bed to another (32,33).The function of pericytes is still unclear; however, morphologic evidence suggests that they may be contractile cells related to vascular smooth muscle. Numerous micro filaments form a continuous plate in the adluminal cytoplasm of their cell body and extend from it into the foot processes, where, in the more distal segments, they fill the cytoplasm to the relative exclusion of other subeellular components. In addition, the foot processes have densities that appear similar to the attachment plaques and dense bodies of smooth muscle cells (29,32,34). The presence of such structures within circumferentially oriented pericyte processes suggests a poten-
The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta are a well-characterized model system for examining the biochemical changes that accompany programmed cell death during development. These giant muscles die during a 30-hr period in response to a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. When the ISMs become committed to die, there are dramatic increases in both ubiquitin expression and ubiquitin-dependent proteolysis. Since the multicatalytic proteinase (MCP) is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its composition and properties. The purified enzyme from whole larval integumentary tissues resembles MCPs isolated from other species with respect to subunit composition and general catalytic properties. However, when MCP was isolated from condemned ISMs, we observed an approximately ninefold increase in proteinase activity compared to MCP from precommitment muscles. This increase in proteolytic activity was correlated with an approximately eightfold increase in the absolute amounts of MCP protein as determined by Western blotting and densitometry. When purified MCP from condemned muscles was examined by two-dimensional polyacrylamide gel electrophoresis, four new subunits that were not detected in the precommitment muscles were present. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. These data are consistent with the hypothesis that when the ISMs become committed to die, more MCP accumulates in cells and new subunits are synthesized that change both the enzymatic properties and the conformation of MCP, which in turn participates in the dramatic proteolysis that accompanies cell death.
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