Use of human vessels and human vascular smooth muscle cells in pharmacology

Relatively limited information is available regarding the mechanisms controlling vasomotricity in human vessels. Isolated vessels obtained from patients undergoing surgery were used to characterize the role of endothelial factors and to study coupling mechanisms between receptors, intracellular calcium, and contraction. However, these investigations are limited by the availability of tissues and many uncontrolled factors. Cultured human vascular cells were also used, were these cells rapidly lose at least some of their differentiated characters. Recently, a human blood vessel equivalent was constructed in vitro from cultured cells, using tissue engineering. This technique allowed us to obtain vessel equivalents containing intima, media, and adventitia layers or tubular media layer only. Contraction and rises in intracellular calcium produced by agonists were studied, indicating that such human vessel equivalents may provide valuable models for pharmacological studies.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na+ decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na+ induced a 6-fold increase in the IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.     

葡萄籽中原花青素对实验动物主动脉收缩和血小板聚集的影响

目的:研究葡萄籽中原花青素(PA)对大鼠离体主动脉平滑肌收缩活动和兔血小板聚集的影响.方法:采用大鼠离体主动脉环灌流方法,记录主动脉环张力变化,观察PA对去甲肾上腺素(NA)和KCl预收缩大鼠离体主动脉平滑肌收缩反应的舒张作用以及对NA量效曲线的影响.比浊法测定兔血小板聚集.结果:PA能明显抑制NA(10-6mol/L)预收缩大鼠离体主动脉环的反应,使NA量效曲线压低,最大反应降低,此作用无内皮依赖性,但对KCl预收缩主动脉环的舒张作用无明显影响,也不影响花生四烯酸(AA),ADP和胶原(collagen)蛋白诱导的兔血小板聚集.结论:PA能对抗NA而不影响KCl诱导的大鼠离体主动脉平滑肌的收缩,不影响兔血小板聚集.     

模拟失重大鼠肠系膜小动脉平滑肌细胞电压依赖性钙离子通道电流的改变

本工作旨在探讨短、中期模拟失重下人鼠肠系膜小动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)电压依赖性钙离子通道(voltage-dependent calcium channels,VDC)功能的改变。以尾部悬吊大鼠模型模拟失重对不同部位血管的影响。采用全细胞膜片钳实验技术,以Ba^2 作为载流子,测定1周及4周模拟失重人鼠肠系膜小动脉VSMCs的VDC电流密度、稳态激活与失活曲线及有关参数,并与对照组结果进行比较。研究表明,本实验所记录到的内向电流主要为钡离子通过长时程VDC(L-VDC)所形成的电流。与对照组相比,1周模拟失重大鼠肠系膜小动脉VSMCs的L-VDc电流密度仪呈降低趋势;但4周模拟失重人鼠肠系膜小动脉VSMCs的L-VDC电流密度则已显著降低。此外,与对照组相比,1、4周模拟失重大鼠肠系膜小动脉VSMCs的膜电容、翻转电位与L-VDC的一些动力学特征值,如通道的开放与关闭速率,通道电流稳态激活与火活曲线及其特征拟合参数V0.5与K的值,均末见有显著改变。结果提示：模拟失重下后身小动脉VSMCs的VDC功能降低可能是模拟失重引起人鼠后身动脉收缩反应性降低及适应性萎缩变化的电生理机制之一。     

Ultrafast Ca wave in cultured vascular smooth muscle cells aligned on a micropatterned surface

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which propagates as Ca²⁺ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca²⁺ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca²⁺ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca²⁺]_i dynamics and membrane potential events along the network of cells. The rate of [Ca²⁺]_i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca²⁺ channels (VOCCs) inhibition suppressed the ultrafast Ca²⁺ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca²⁺ entry from the external medium through VOCCs, modeled as an ultrafast Ca²⁺ wave. This wave may trigger and drive slower Ca²⁺ waves observed ex vivo and in vivo.     

Mechanism of inhibition by alloxan of ATP-driven calcium transport by vascular smooth muscle microsomes

The directin vitro effects of alloxan on the Ca²⁺ handling by microsomal membranes isolated from dog mesenteric arteries were investigated. Preincubation of the vascular muscle microsomal membranes with alloxan showed a suppressive effect on both binding of Ca²⁺ (in the absence of ATP) and ATP-driven Ca²⁺ transport. Such an inhibition was time dependent, dose dependent, and temperature dependent. ATP-driven Ca²⁺ transport was much more susceptible to the inhibitory action of alloxan than Ca²⁺ binding under all experimental conditions examined. Alloxan inhibited ATP-driven Ca²⁺ transport at a comparable level over the entire period of Ca²⁺ uptake, but had no significant effect on the efflux of Ca²⁺ from preloaded microsomal membranes. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca²⁺ transport via its action on the Ca-pump protein rather than the membrane permeability to Ca²⁺. Catalase and mannitol but not superoxide dismutase partially protected against such as inhibition by alloxan. The possible involvement of H₂O₂ mediating the inhibitory action of alloxan was further supported by the finding of a similarin vitro inhibitory effect of H₂O₂ on the ATP-driven Ca²⁺ transport by the vascular smooth muscle microsomes.     

影响主动脉平滑肌细胞迁移的因素

平滑肌细胞(vascular smooth muscle cell,VSMC)的迁移对血管发育、动脉粥样硬化和术后再狭窄等起到关键性的作用。主要从激发VSMC迁移的关键炎性细胞因子、细胞间相互作用的核心成员、microRNA、细胞骨架和上述各因素的迁移信号通路这几方面来综述VSMC的迁移。     

Cloning of an apamin binding protein of vascular smooth muscle

The receptor for the bee venom derived neurotoxin, apamin, is widely believed to be an integral component of the small conductance calcium-activated potassium channel in many excitable cells. By affinity chromatography on immobilized apamin, a 78 kD apamin binding protein of the bovine brain synaptosomes was isolated. Antibodies were elicited against this protein and used to clone a cDNA from a porcine vascular smooth muscle expression library. This gene (Kcal 1.8) codes for a 438 amino protein with four potential transmembrane domains, one putative calcium binding site, a protein kinase C phosphorylation site, and a leucine zipper motif. Kcal 1.8 encoded protein has no significant sequence homologies with any known ion channels or receptors. Kcal 1.8 is likely to encode a protein associated with the small conductance calcium-activated potassium channel in vascular smooth muscle.     

Sorting of murine vascular smooth muscle cells during wound healing in the chicken chorioallantoic membrane

The vascular wall is built up of a heterogeneous population of smooth muscle cells, which exhibit not only morphological distinctions but also important differences in the composition of their structural and contractile proteins. "Epithelioid" smooth muscle cells correspond to an intimal-like type and display features associated with immaturity, whereas "spindle-shaped" cells closely resemble the more typical medial smooth muscle population. We have investigated the integration of these two cell types into the vascular architecture of an in vivo wound-healing model. Stably transfected with the beta-galactosidase gene, intima- and media-like cells were injected intravenously into the chicken chorioallantoic membrane, within which superficial foci of granulation tissue had been created by thermal or chemical injury. At 24 to 72 h after injection, cells had honed in on the lesion sites and were observed in juxtaposition to the endothelial lining of the capillaries. They began to deposit laminin, thereby indicating an impending role in the formation of the vascular wall. Intima- and media-like smooth muscle cells did not differ in their capacity to associate with capillaries, and, in so doing, their biochemical lineage characteristics became indistinguishable from one another. However, intima-like cells also penetrated the adventitial and medial layers of arteries. These findings reveal vascular smooth muscle cells to possess an extraordinary degree of plasticity, being able to adapt flexibly to changes in functional demands.     

The calpain—calpastatin system in vascular smooth muscle

Vascular smooth muscle contains large amounts of the Ca²⁺-dependent protease calpain II. In this study, we compared bovine aortic muscle (muscle phenotype) to cultured bovine aortic cells of smooth muscle origin (modulated phenotype) with respect to major constituents of the calpain—calpastatin system. Bovine aortic muscle contained only calpain II by activity measurements, Western blot of tissue extracts and Northern blot of poly(A)⁺ RNA. On the other hand, using the same methodologies, both calpains I and II as well as the 110 kDa inhibitor protein, calpastatin, were identified in cultured bovine aortic cells of smooth muscle origin. We conclude that the phenotypic state of smooth muscle cells is associated with differential expression of major components of the calpain—calpastatin system. Moreover, bovine aortic muscle is the only tissue identified to date that contains calpain II exclusively.     

四周模拟失重大鼠后身动脉平滑肌细胞钾电流的改变

本文采用全细胞膜片钳方法观察4周尾部悬吊大鼠（tail-suspended rats,SUS)隐动脉及肠系膜的动脉第2-6级动脉分支血管平滑肌细胞（vascular smooth muscle cells,VSMCs)钾电流密度的变化,结果表明：SUS大鼠后身动脉VSMCs的静息电位（RP）较对照大鼠（CON）后身动脉VSMCs的RP更负,SUS组隐动脉和肠系膜小鼠后身动脉VSMCs的静息电位（RP）较对照大鼠（CON）后身动脉VSMCs的RP更负,SUS组隐动脉和肠系膜小动脉VSMCs的全细胞钾电流密度较CON组显著增加,其中,SUS组的隐动脉和肠系膜小动脉VSMCs的大电导钙激活钙离子通道（BKca)和电压激活钾离子通道（Kv)电流密度较CON组的BKca和Kv电流密度均显著增加,以上结果提示,VSMCs的超极化及进一步引起的通过电压依赖性钙离子通道的钙内流减少可能是模拟失重引起后身动脉反应性降低的电生理机制之一。     

Compartmentalized coculture of porcine arterial endothelial and smooth muscle cells on a microporous membrane

Summary Endothelial and smooth muscle cells were harvested from porcine pulmonary arteries and grown to two passages from primary culture in serum-containing medium. Thereafter, the cells were plated on the opposite sides of a microporous poly-(ethylene terephthalate) membrane and cultivated in a chemically defined, serum-free medium. The membrane with pores of 1 μm diameter allowed the passage of molecules and the extension of cell processes, while maintaining separate homogeneous cell populations. Pores of 3 μm diameter permitted the crossing of smooth muscle cells through the membrane. The coating of the polymer with constituents of the extracellular matrix optimized cell adhesion. Morphological analysis of the model showed typical cobblestone pattern and ultrastructure of endothelial cells, which lost rapidly the expression of von Willebrand factor but kept that of angiotensin-converting enzyme. Smooth muscle cells were spindle shaped and specific α-actin was revealed by immunochemistry and quantitated by enzyme-linked immunosorbent assay (ELISA). Their ultrastructure featured an intermediate contractile-synthetic phenotype. Permeability studies to different molecules showed a marked reduction of the albumin clearance. Finally, in coculture in the presence of endothelial cells, the smooth muscle cells proliferation was increased, whereas it was not the case in autologous cocultures. In conclusion, such a coculture model may help to a better understanding of the interactions between endothelial and smooth muscle cells that may be important in the pathogenesis of vascular diseases.     

Properties of potassium activatedp-nitrophenyl phosphatase of plasma membranes isolated from rat stomach muscle

The plasma membrane-enriched fraction isolated from smooth muscle of rat gastric fundus was found to contain a substantial level of potassium-stimulatedp-nitrophenylphosphatase activity (K-pNPPase), and its subcellular distribution closely resembled that of other plasma membrane enzyme markers. The kinetic profile of K-pNPPase and its sensitivity toward ouabain and vanadate confirmed the identification of this activity with the partial reaction of the sodium pump. The specific activity of K-pNPPase and its sensitivity to ouabain was significantly increased in the presence of saponin, indicating that part of this activity is latent when assayed on native membrane preparation. K-pNNPase was sensitive to the presence of calcium ions in the assay medium. The Ca²⁺-inhibition of K-pNNPase was accompanied by increased sensitivity of the enzyme to ouabain. On the other hand, calmodulin and Ca antagonists had no effect on K-pNPPase activity nor its sensitivity to calcium.     

Hyperosmolality-induced abnormal patterns of calcium mobilization in smooth muscle cells from non-diabetic and diabetic rats

Hyperglycemia and/or hyperosmolality may disturb calcium homeostasis in vascular smooth muscle cells (SMCs), leading to altered vascular contractility in diabetes. To test this hypothesis, the KCl induced increases in [Ca²⁺]_i in primarily cultured vascular SMCs exposed to different concentrations of glucose were examined. With glucose concentration in solutions kept at 5.5 mM, KCl induced a fast increase in [Ca²⁺]_i which then slowly declined (type 1 response) in 83% of SMCs from non-diabetic rats. In 9% of non-diabetic SMCs KCl induced a slow increase in [Ca²⁺]_i (type 2 response). Interestingly, under the same culture conditions KCl induced type 1 and type 2 responses in 47 and 35% of SMCs from diabetic rats. When SMCs from non-diabetic or diabetic rats were cultured in 36 mM glucose, KCl induced a fast increase in [Ca²⁺]_i which, however, maintained at a high level (type 3 response). The sustained level of [Ca²⁺]_i in the presence of KCl was significantly higher in cells cultured with 36 mM glucose than that in non-diabetic cells cultured with 5.5 mM glucose. Furthermore, the hyperglycemia-induced alterations in calcium mobilization were similarly observed in cells cultured in high concentration of mannitol (30.5 mM) or L-glucose, indicating that hyperosmolality was mainly responsible for the abnormal calcium mobilization in diabetic SMCs.     

Crystallizing nanoparticles derived from vascular smooth muscle cells contain the calcification inhibitor osteoprotegerin

Osteoprotegerin (OPG), a member of the TNF receptor superfamily, was initially found to modulate bone mass by blocking osteoclast maturation and function. Rodent models have also revealed a role for OPG as an inhibitor of vascular calcification. However, the precise mode of how OPG blocks mineralization is unclear. In this study, OPG was found in an in vitro assay to significantly inhibit calcification of vascular smooth muscle cells (VSMC) induced by high calcium/phosphate (Ca/P) treatment (p = 0.0063), although this effect was blunted at high OPG concentrations. By confocal microscopy, OPG was detected in VSMC in the Golgi, the same localization seen in osteoblasts, which express OPG in bone. Treatment of VSMC by minerals (Ca, P, or both) induced OPG mRNA expression as assessed by real-time quantitative PCR, and VSMC derived from atherosclerotic plaque material also exhibited higher OPG expression as compared to control cells (p < 0.05). Furthermore, OPG was detected by Western blotting in matrix vesicles (MV), nanoparticles that are released by VSMC with the capacity to nucleate mineral. In atherosclerotic arteries, OPG colocalized immunohistochemically with annexin VI, a calcium-dependent membrane and phospholipid binding protein found in MV. Thus, the calcification inhibitor OPG is contained in crystallizing MV and has a biphasic effect on VSMC: physiologic concentrations inhibit calcification, whereas high concentrations commonly seen in patients with vascular disease have no effect. Like other calcification inhibitors, OPG may be specifically loaded into these nanoparticles to be deposited at remote sites, where it acts to inhibit calcification.     

Gene expression profile of butyrate-inhibited vascular smooth muscle cell proliferation

Excessive proliferation of vascular smooth muscle cells (VSMCs) is a critical element in the development of several vascular pathologies, particularly in atherosclerosis and in restenosis due to angioplasty. We have shown that butyrate, a powerful antiproliferative agent, a strong promoter of cell differentiation and an inducer of apoptosis inhibits VSMC proliferation at physiological concentrations with no cytotoxicity. In the present study, we have used cDNA array technology to unravel the molecular basis of the antiproliferative effect of butyrate on VSMCs. To assess the involvement of gene expression in butyrate-inhibited VSMC proliferation, proliferating VSMCs were exposed to 5 mmol/1 butyrate 1 through 5 days after plating. Expression profiles of 1,176 genes representing different functional classes in untreated control and butyrate treated VSMCs were compared. A total of 111 genes exhibiting moderate (2.0–5.0 fold to strong (> 5.0 fold) differential expression were identified. Analysis of these genes indicates that butyrate treatment mainly alters the expression of four different functional classes of genes, which include: 43 genes implicated in cell growth and differentiation, 13 genes related to stress response, 11 genes associated with vascular function and 8 genes normally present in neuronal cells. Examination of differentially expressed cell growth and differentiation related genes indicate that butyrate-inhibited VSMC proliferation appears to involve down-regulation of genes that encode several positive regulators of cell growth and up-regulation of some negative regulators of growth or differentiation inducers. Some of the down-regulated genes include proliferating cell nuclear antigen (PCNA), retinoblastoma susceptibility related protein p130 (pRb), cell division control protein 2 homolog (cdc2), cyclin B1, cell division control protein 20 homolog (p55cdc), high mobility group (HMG) 1 and 2 and several others. Whereas the up-regulated genes include cyclin D1, p21WAF1, p14INK4B/p15INK5B, Clusterin, inhibitor of DNA binding 1 (ID1) and others. On the other hand, butyrate-responsive stress-related genes include some of the members of heat shock protein (HSP), glutathione-s-transferase (GST), and glutathione peroxidase (GSH-PXs) and cytochrome P450 (CYP) families. Additionally, several genes related to vascular and neuronal function are also responsive to butyrate treatment. Although involvement of genes that encode stress response, vascular and neuronal functional proteins in cell proliferation is not clear, cDNA expression array data appear to suggest that they may play a role in the regulation of cell proliferation. However, cDNA expression profiles indicate that butyrate-inhibited VSMC proliferation involves combined action of a proportionally large number of both positive and negative regulators of growth, which ultimately causes growth arrest of VSMCs. Furthermore, these butyrate-induced differential gene expression changes are not only consistent with the antiproliferative effect of butyrate but are also in agreement with the roles that these gene products play in cell proliferation.     

Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle

This report demonstrates that the commonly used anesthetic agent, pentobarbital sodium, in concentrations of 1 · 10^?4 to 2 · 10^?3 M inhibits calcium (Ca²⁺) uptake in both rat aortic and portal venous smooth muscle. The data indicate that total exchangeable Ca²⁺ in portal vein is reduced by about 15% in 1 · 10^?4 M pentobarbital sodium, while the intracellular exchangeable Ca²⁺ is reduced by 24%. On the other hand, in aortic smooth muscle, while 5–20 · 10^?4 M pentobarbital sodium reduces total exchangeable Ca²⁺ by about 15%, intracellular Ca²⁺ is reduced by 22% in 5 · 10^?4 M pentobarbital sodium and by 38% in 2 · 10^?3 M pentobarbital sodium. The present studies thus reveal that concentrations of pentobarbital sodium known to be present during induction of surgical anesthesia can exert significant inhibitory effects on exchangeability and transmembrane movement of Ca²⁺ in at least two different types of blood vessels.     

Large scale preparation of rabbit aortic smooth muscle cells for use in calcium uptake studies

Summary Rabbit aortic smooth muscle cells were prepared by enzymatic digestion of the aortic smooth muscle layer. The cells were subcultured up to Passage 22 starting from a cryogenically preserved stock (approximately 10¹⁰cells, Passage 8) and characterized morphologically and for⁴⁵Ca⁺⁺ uptake. Microscopically the cells demonstrated the characteristics of vascular smooth muscle cells.⁴⁵Ca⁺⁺ uptake by the cells plated on tissue culture flasks (25 cm²) was determined at 25°C in physiological salt solution (PSS) containing⁴⁵Ca⁺⁺ in low (5 mM) or high (50mM) KCl concentrations. At the end of the incubation period (0 to 30 min), PSS was aspirated and the cells quickly washed, digested with 0.5N NaOH, and counted for⁴⁵Ca⁺⁺. High K⁺ increased the⁴⁵Ca⁺⁺ uptake by 100% or more compared to the low K⁺ uptake of⁴⁵Ca⁺⁺. This K⁺-induced⁴⁵Ca⁺⁺ uptake was eliminated in osmotically shocked cells, and inhibited by nifedipine, verapamil, and diltiazem, in a dose-dependent manner. The extent of⁴⁵Ca⁺⁺ uptake and the inhibitory activity of nifedipine were retained up to Passage 22. It is concluded that the developed methodology for scaled-up cultures of rabbit aortic smooth muscle cells provides morphologically intact and biochemically functioning cells suitable for calcium channel studies.     

A morphometric study of vascular smooth muscle cells in culture

Summary Cultured arterial smooth muscle cells derived from different times in culture, different passages, and different species were evaluated by a combination of transmission electron microscopy and morphometry. The morphometric studies focused on point counting and monitored the following cellular components: lysosomes, myofilaments, mitochondria, ribosomes, and rough endoplasmic reticulum (RER). Percent volume composition values for the organelles involved in protein synthesis, namely ribosomes and RER, show significant fluctuations with time. Consistent with these observations, the cells showed increasing myofilaments during the early weeks in culture, which subsequently decreased significantly. The data also indicate that rabbit cells in culture may become synthetically quiescent with time and the distribution of cellular components is altered with each succeeding passage. Cultured calf (bovine) cells exhibit similar activity periods compared to rabbit but show a significantly higher lysosomal and lower myofilament content than rabbit. Calf cells could not be maintained for longer than 21 days in the absence of ascorbate, whereas ascorbate affects the ultrastructure of rabbit cells less dramatically. Age, passage, and donor, among others, are important considerations for studying in vitro smooth muscle cells. With proper morphologic and morphometric monitoring, these smooth muscle cell culture systems can be important tools in the study of aging or pathologic processes, or both. This work was presented as partial fulfillment for the degree of Ph.D. This work was supported by National Institutes of Health Grants HL-13262, HL-19717, and AG-00001.