Contribution of chondroitin-dermatan sulfate-containing proteoglycans to the function of rat mesenteric arteries.

Proteoglycans are an important nonfibrous matrix component of the arterial wall. Direct evidence for their role in resistance-sized arteries is lacking, although they likely have an important role in coordinating and regulating vessel behavior, presumably via interactions of their glycosaminoglycan chains or core proteins with other matrix molecules and/or the smooth muscle cell surface. The purpose of this study was to determine whether the removal of specific glycosaminoglycan chains from proteoglycans in resistance-sized mesenteric arteries would change the mechanical properties of the arterial wall, thereby affecting their functional behavior. The major finding of the study was that 65% removal of chondroitin-dermatan sulfate-containing glycosaminoglycans from the arterial wall increased vascular wall stiffness and altered the myogenic behavior of the artery. The significant alterations in myogenic behavior associated with changes in passive mechanics following partial glycosaminoglycan chain removal support our hypothesis that chondroitin-dermatan sulfate-containing proteoglycans contribute significantly to the functional behavior of resistance arteries. We speculate that these alterations are the result of changes in stress transfer between collagen fibrils and/or stress transfer between cells and collagen fibrils under applied pressure.

Tunica media remodeling in mesenteric arteries of hypertensive rats.

BACKGROUND: The tunica media of the vascular wall is a composite material comprised of smooth muscle cells and fibrous and nonfibrous matrix proteins. METHODS: Using morphometric techniques, this study quantifies the cell and matrix composition of normotensive (Wistar-Kyoto) and spontaneously hypertensive rat mesenteric arteries. RESULTS: The data show that the superior mesenteric artery (SMA) and small mesenteric arteries are different in matrix composition, cell-to-matrix ratio, and cellular dense body content. Compared with normotensive arteries, hypertensive arteries have less basement membrane but more collagen and extracellular matrix ground substance. SMA from hypertensive rats has about 30% less elastin than does normotensive artery. In contrast, the elastin content of small arteries of both strains was about the same and was less than 4% of the matrix area. Except in hypertensive SMA, membrane dense bodies occupy 7-10% of the cell area and more than 10 times the area occupied by cytoplasmic dense bodies. In contrast, cells from hypertensive SMA have about half the membrane dense body area of the normotensive cells. A decreased proportion of dense bodies in the hypertensive SMA is consistent with the "partial detachment" of these cells from the matrix. CONCLUSIONS: These results are consistent with both cellular and matrix "remodeling" in diseased vessels in response to continuous, long-term elevated blood pressure.

Composition in situ and in vitro of vascular smooth muscle laminin in the rat.

Vascular smooth muscle cells are surrounded by a basal lamina containing an array of macromolecules: included among these are the laminins, a family of oligomeric glycoproteins composed of subunits encoded by different genes. In this study, we have used monoclonal antibodies to several of these subunits, including S-laminin, laminin B2, and laminin B1, to study these proteins in tail artery, superior mesenteric artery, and aorta of rats. In situ, immunostaining for the B2 and S chains was present in the basal lamina, between the smooth muscle cells, throughout the tunica media. In contrast, B1 chain immunostaining was concentrated around cells in the inner media. To investigate whether smooth muscle cells can produce S-laminin, laminin B2, and laminin B1, smooth muscle cells from the superior mesenteric artery were grown in culture and laminin subunit expression determined. In early culture (4 days), immunostaining showed abundant laminin B2 and less B1 synthesis and incorporation into the matrix. Staining for S-laminin was even less intense than for B1 and was localized to areas where cells were densely packed. The same pattern of S-laminin immunostaining was seen during early culture in cells grown on fibronectin, type IV collagen, or gelatin. Immunoblotting detected S-laminin in the conditioned medium from early cultured cells. In later culture (12 days), S-laminin incorporation into the matrix increased markedly compared to incorporation at 4 days. At this time, cells are much more densely packed and multilayered with extensive matrix accumulation. Cyclical stretching of cells in vitro did not increase immunostaining for S-laminin. Together these data show that S-laminin is a component of the arterial media in situ and that in vitro S-laminin is synthesized by smooth muscle cells. Increased incorporation of S-laminin into the matrix in later culture correlates with the presence of a more extensive matrix, suggesting that matrix organization may be critical to S-laminin incorporation.

Isolation and culture of rat superior mesenteric artery smooth muscle cells.

The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.

Three-dimensional characterization of dense bodies in contracted and relaxed mesenteric artery smooth muscle cells.

We have previously shown that dense bodies are not the static planar simple ovoidal structures they appear to be in thin sections. In this report, we present three-dimensional reconstructions from consecutive serial thin sections through shortened and non-shortened large mesenteric artery cells. Profiles of the cell surface, membrane dense bodies, and cytoplasmic dense bodies were reconstructed from consecutive thin sections and the distribution, size, shape, and spatial relationships among these components was examined. Within the cell, membrane dense bodies are numerous and occupy approximately 10% of the cell volume. Membrane dense bodies can attach to the cell surface laterally, obliquely or normally. An individual membrane dense body can be continuous over more than 2 microns of cell depth and can change shape throughout its depth. On cell shortening, many membrane dense bodies assume a crenated shape. Compared to membrane dense bodies, cytoplasmic dense bodies are smaller in all dimensions and occupy about 2% of the cell volume. In shortened cells, cytoplasmic dense bodies appear to cluster into groups. This redistribution of cytoplasmic dense bodies may be related to the reorganization of contractile units when the cell shortens.

Proteoglycan synthesis in normotensive and spontaneously hypertensive rat arteries in vitro.

Proteoglycans (PGs) were analyzed and compared in the media of the thoracic aorta, abdominal aorta, left carotid artery and superior mesenteric artery of age-matched Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Two ages were examined; 10 week old, during the development of hypertension and 28 week old, when hypertension is well established in the SHR. Large chondroitin sulfate PG, large heparan sulfate PG and biglycan (PGI) and decorin (PGII) small PGs were identified. Biglycan was the predominant small PG found in all arteries. Newly synthesized PGs were labelled in vitro with 35SO4 for quantitation. The synthesis of large and small PGs was similar in the media of the thoracic aorta, abdominal aorta, left carotid artery, and superior mesenteric artery. The large to small ratio value, a measure of the artery PG composition, was also similar among the four arteries but was highest in the mesenteric artery. In both WKY and SHR arteries there was significantly decreased PG synthesis in the 28-week old compared to 10-week old animals. This was especially true for large PG. Hypertensive changes in PG synthesis were seen mainly in the carotid artery. In this artery, synthesis of both large and small PG was increased in the SHR, at both ages. The ratio of large to small PG was not significantly different between SHR and WKY arteries. We conclude that 28-week old WKY and SHR rat arteries synthesize less large and small PG than 10-week old arteries. The most prominent change seen in hypertensive rats is an increase in PG synthesis in the carotid artery.

Organization of cells and extracellular matrix in mesenteric arteries of spontaneously hypertensive rats.

Biochemical studies have been used to assess the quantitative changes in elastin and collagen in hypertensive vs. normotensive arteries. However, the relative distribution and organization of these fibrous proteins is likely to be equal in importance to their absolute amounts. In this study we have used scanning electron microscopy in association with selective digestion techniques to assess the organization of cellular and extracellular components of the tunica media of mesenteric arteries of spontaneously hypertensive rats. Superior and small mesenteric arteries were digested with acid, alkali, or bleach to exposure cells, collagen, or collagen and elastin, respectively. We observed that hypertension does not cause a qualitative change in the 3-dimensional arrangement of cells, collagen, or elastin in spontaneously hypertensive arteries when compared to normotensive arteries. However, cells in the superior artery are significantly different in overall shape and surface features when compared to cells of small arteries. These differences in surface morphology of cells are present in hypertensive and normotensive vessels and suggest that superior and small mesenteric artery cells transmit load to the isotropic matrix in different ways. In the elasto-muscular superior artery, force is transmitted across digitations throughout the cell surface. In the muscular small artery, force is transmitted across the tapered, smooth cell surface.

Organization of rat mesenteric artery after removal of cells of extracellular matrix components.

Rat mesenteric arteries, perfusion fixed in relaxed or contracted conditions, were digested with acid and elastase, bleach (sodium hypochlorite), or alkali to selectively remove collagen, elastin, or cells. Scanning electron microscopy was used to study the three-dimensional organization of the remaining cells or extracellular components. Smooth muscle cells of the tunica media were elongated and circumferentially oriented. Superior mesenteric artery cells had an irregular surface with numerous projections and some ends were forked. Small mesenteric artery cells were spindle shaped with longitudinal surface ridges, and showed extensive corrugations upon contraction. Elastin was present both as laminae and as an interconnected fibrous meshwork. Collagen was arranged in an irregular network of individual fibrils and small bundles of fibrils that formed nests around the cells in both arteries. This irregular arrangement persisted, with no apparent reordering or loss of order, upon contraction. The lack of an ordered arrangement or specialized organization at the cell ends suggests mechanical coupling of the cells to elastin or collagen throughout the length of the cell, allowing for force transmission in a number of directions. The tunica media is thus a "composite" material consisting of cells, elastin, and collagen. The isotropic network of fibers is well suited for transmitting the shearing forces placed on it by contraction of smooth muscle cells and by pressure-induced loading.

Three-dimensional structure of dense bodies in rabbit renal artery smooth muscle.

In this report, we present a three-dimensional computer assisted reconstruction study from serial thin sections through a rabbit renal artery smooth muscle cell. In a series of 32 consecutive thin (100-nm) sections, one longitudinally oriented cell was followed and photographed in alternating sections. The profiles of the cell surface and dense bodies were reconstructed from these 16 planes and the distribution, size, shape, and spatial relationships among these components was examined. The reconstructed images showed that the cell decreases in diameter from its widest region in the center to the two ends in a step-wise taper. Within the cell, dense bodies are numerous. Relative to the cell axes, a membrane associated dense body (MDB) can be less than or equal to 3.5 microns long, 0.25 micron wide, and may extend up to 2 microns in depth. While the MDB profile in one section may be aligned with the long axis of the cell, in an adjacent section the same dense body may appear almost circular or wedge shaped. The same is true of cytoplasmic dense bodies (CDBs). Compared with MDBs, CDBs are smaller in all dimensions. Some, but not all, CDBs line up in strings oriented with the long axis of the cell. The continuity of dense bodies over considerable cell depth and their change in shape may have important implications for integration of contractile activity and for transmitting passive tension to the extracellular matrix.

Effects of temperature and buffer composition on calcium sequestration by sarcoplasmic reticulum and plasma membrane of rabbit renal artery.

45Ca electron microscopic autoradiography was used to examine the effects of buffer composition and temperature on the distribution of calcium in rabbit renal artery smooth muscle cells. The results show that the relative distribution of calcium is dependent on both the buffer used (Tris or Krebs) and the temperature of the bathing solution (25 degrees C or 34 degrees C). Krebs buffer at 34 degrees C gave the highest relative activity in the plasma membrane, sarcoplasmic reticulum, and mitochondria. Buffer and temperature had little effect on the relative activity of the nucleus or cytoplasm. Next, we identified the cellular sites of calcium accumulation after 5, 15, 30, or 60 min exposure to 45Ca in Krebs buffer at 34 degrees C. The results show that sarcoplasmic reticulum and plasma membrane are the primary sites of calcium accumulation during influx into these cells. Although the amount of 45Ca in the cell continues to increase with longer exposure, the relative distribution of calcium is essentially the same after 5 or 60 min. The data also indicate that the relative activity of plasma membrane + sarcoplasmic reticulum (a combination site that includes sarcoplasmic reticulum within a mean distance of 275 nm of the plasma membrane) is similar to the membrane alone and is lower than the sarcoplasmic reticulum alone.

Effect of calcium channel antagonists on calcium uptake and release by isolated rat cardiac mitochondria.

The effects of calcium channel antagonists on Ca2+ uptake and Na+-induced Ca2+ release were studied in isolated rat cardiac mitochondria. Diltiazem, nitrendipine and nimodipine were more effective inhibitors of Na+-induced Ca2+ release (IC50 = 19-100 microM) than of Ca2+ uptake (IC50 = 0.2-1 mM). Nitrendipine and nimodipine had virtually identical IC50 values for inhibiting Ca2+ uptake, but nitrendipine was 3-4 times more potent than nimodipine at inhibiting Na+-induced Ca2+ release. If these calcium channel antagonists achieve intracellular concentrations in the range of 10(-5)-10(-4) M, our results suggest that calcium channel antagonists would preferentially inhibit mitochondrial calcium release more than mitochondrial calcium uptake.

Proteoglycan in fast-frozen, freeze-dried, plastic-embedded rabbit arteries.

In contrast to glutaraldehyde-fixed vascular tissue with or without staining with cationic dye, the nonfibrous extracellular matrix of fast-frozen, freeze-dried rabbit aorta and renal artery contained a continuous reticulum of fine filaments, closely associated with collagen, elastin, and smooth muscle cells. Three morphologically distinct types of filament were distinguished; one type was selectively sensitive to chondroitinase ABC degradation, and therefore contains chondroitin and/or dermatan sulfate. The remaining filaments of the reticulum may represent the protein core of the proteoglycan monomer, and the hyaluronic acid backbone of the aggregate. Filaments associated with the surface of smooth muscle cells were usually linked to a continuous filament parallel to the cell surface, which was degraded by heparitinase and therefore contains heparan sulfate. The filaments linked directly to the cell surface were not degraded by either enzyme. The preservation of PG in fast-frozen material provides a significant improvement over that obtained by any presently available technique.

Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue.

Immunoelectron microscopic labeling of calsequestrin on ultra-thin sections of rat ventricular muscle prepared by quick-freezing, freeze-drying, and direct embedding in Lowicryl K4M was compared to that observed on ultra-thin sections prepared by chemical fixation, dehydration in ethanol, and embedding in Lowicryl K4M. Brightfield electron microscopic imaging of cryofixed, freeze-dried, osmicated, and Spurr-embedded rat ventricular tissue showed that the sarcoplasmic reticulum was very well preserved by cryofixation and freeze-drying. Therefore, the four structurally distinct regions of the sarcoplasmic reticulum (i.e., the network SR, the junctional SR, the corbular SR, and the cisternal SR) were easily identified even when myofibrils were less than optimally preserved. As previously shown by immunoelectron microscopic labeling of ultra-thin frozen sections of chemically fixed tissue, calsequestrin was confined to the lumen of the junctional SR and of a specialized non-junctional (corbular) SR, and was absent from the lumen of network SR in cryofixed, freeze-dried, Lowicryl-embedded myocardial tissue. In addition, a considerable amount of calsequestrin was also present in the lumen of a different specialized region of the non-junctional SR, called the cisternal sarcoplasmic reticulum. By contrast, relocation of calsequestrin to the lumen of the network SR was observed to a variable degree in chemically fixed, ethanol-dehydrated, and Lowicryl-embedded tissue. We conclude that tissue preparation by cryofixation, freeze-drying, and direct embedding in Lowicryl K4M for immunoelectron microscopic localization of diffusible proteins, such as calsequestrin, is far superior to that obtained by chemical fixation, ethanol dehydration, and embedding in Lowicryl K4M.

Localization of calcium in vas deferens using 45Ca EM autoradiography: relationship to species and the effect of 45Ca removal.

The distribution of calcium was determined in the vas deferens of the guinea pig using 45Ca electron microscopic autoradiography of rapidly frozen, freeze-dried, and embedded tissue. A selective accumulation of calcium at the plasma membrane and SR was observed in vas deferens that had been incubated in 45Ca for 65-85 min prior to rapid freezing. Rinsing the tissue in nonradioactive calcium for 6 min prior to rapid freezing significantly altered the distribution of calcium among the plasma membrane, mitochondria, and cytoplasmic matrix. The influence of species on the observed distribution of calcium was also examined. The distribution of calcium in the guinea pig vas deferens was not significantly different from that in the rabbit vas deferens when the tissues were prepared under identical conditions.

Cellular redistribution of 45Ca in rabbit renal artery determined by electron microscopic autoradiography: changes in response to K+-induced depolarization and nitrendipine.

Redistribution of Ca++ in response to K+-induced depolarization and/or nitrendipine (10(-6) M) was studied in isolated rabbit renal arteries using 1) isometric tension measurements, 2) 45Ca uptake measurements and 3) 45Ca electron microscopic autoradiography. Renal artery rings developed a mean tension of 1.67 +/- 0.30 g in response to high K+. Preincubation with 10(-6) M nitrendipine for 1 hr inhibited 80% of the initial portion and 100% of the sustained portion of the K+-induced contraction, without affecting rest tension. Using the modified lanthanum technique, cellular uptake of 45Ca increased 25% in muscles exposed to K+-substituted solution compared to control muscles (P less than .001). Preincubation with nitrendipine for 1 hr inhibited the K+-induced increase, whereas exposure to nitrendipine alone decreased 45Ca uptake compared to control muscles (P less than .001). Electron microscopic autoradiography of control renal arteries showed that relative 45Ca activities for the plasma membrane (PM), sarcoplasmic reticulum (SR) and mitochondria were higher than the cytoplasmic 45Ca activity, whereas activities for the nucleus were similar to that for the cytoplasm. Exposure to high K+ solution substantially decreased 45Ca activities of both the PM and SR (P less than .001), but changes in relative activities of other sites were insignificant. Muscles exposed to nitrendipine alone or nitrendipine plus high K+ had SR and PM activities intermediate with values from control and high K+ groups. Thus, Ca++ is released from both the SR and PM during exposure to high K+ and this Ca++ may contribute to the Ca++ pool involved in a depolarization-induced contraction.

Statistical significance tests for autoradiographic data.

The purpose of this paper is to develop statistical methods that take radiation spread into account in analyzing data from different autoradiographic experiments. The method uses the probability circle analysis of Salpeter and McHenry (1973) to obtain the probable source of each radioactive emission and the circle and point counting method of Williams (1969) to estimate the relative area occupied by each cellular site. Two levels of analysis are presented. The first level of analysis is concerned with estimating relative activities and standard errors for cellular items that are larger than the probability circle. The second level of analysis involves estimating relative activities and standard errors for cellular sites that are smaller than the probability circle and are therefore observed in circles containing another item such as cytoplasmic matrix. Two different tests of hypotheses are discussed. The first null hypothesis is that the radioactivity is randomly distributed among the cellular sites. The second null hypothesis is that there is no difference between two different treatments in the relative activities for a given site.

The cellular distribution of calcium in freeze-dried rabbit vas deferens using EM autoradiography.

The role of calcium in initiating smooth muscle contraction is widely accepted. The sources of this calcium are thought to be located both intracellularly and extracellularly. We have recently developed a method by which the cellular localization of calcium in smooth muscle can be determined. This method involves exposing the tissue to 45Ca, rapidly freezing and vacuum dehydrating the tissue, and preparing the tissue for electron microscopic autoradiography (EM ARG). In the present study the distribution of calcium in control and potassium-contracted tissue of the rabbit vas deferens was compared. No significant differences in distribution were observed in the two treatments. This finding provides morphological support for the hypothesis that the calcium used in potassium-induced contraction is primarily of extracellular origin. In addition, significant sequestration by intracellular organelles does not occur during a potassium contraction. In other experiments, the effect of rinsing tissue in cold calcium prior to freezing was investigated. From these data is appears that calcium is removed from the cytoplasmic matrix, plasma membrane, and organelles in a nonuniform manner. Further investigation into these findings should enable us to characterize more precisely the intracellular redistribution of calcium that occurs as a result of a variety of physiological manipulations.