Structural and Functional Differences Between Porcine Aorta and Vena Cava.

Elastin and collagen fibers are the major load-bearing extracellular matrix (ECM) constituents of the vascular wall. Arteries function differently than veins in the circulatory system; however as a result from several treatment options, veins are subjected to sudden elevated arterial pressure. It is thus important to recognize the fundamental structure and function differences between a vein and an artery. Our research compared the relationship between biaxial mechanical function and ECM structure of porcine thoracic aorta and inferior vena cava. Our study suggests that aorta contains slightly more elastin than collagen due to the cyclical extensibility, but vena cava contains almost four times more collagen than elastin to maintain integrity. Furthermore, multiphoton imaging of vena cava showed longitudinally oriented elastin and circumferentially oriented collagen that is recruited at supraphysiologic stress, but low levels of strain. However in aorta, elastin is distributed uniformly, and the primarily circumferentially oriented collagen is recruited at higher levels of strain than vena cava. These structural observations support the functional finding that vena cava is highly anisotropic with the longitude being more compliant and the circumference stiffening substantially at low levels of strain. Overall, our research demonstrates that fiber distributions and recruitment should be considered in addition to relative collagen and elastin contents. Also, the importance of accounting for the structural and functional differences between arteries and veins should be taken into account when considering disease treatment options.

Generation and characterization of feline arterial and venous endothelial cell lines for the study of the vascular endothelium.

BACKGROUND: The in vitro culture of endothelial cells (ECs) is an indispensable tool for studying the role of the endothelium in physical and pathological conditions. Primary ECs, however, have a restricted proliferative lifespan which hampers their use in long-term studies. The need for standardized experimental conditions to obtain relevant and reproducible results has increased the demand for well-characterized, continuous EC lines that retain the phenotypic and functional characteristics of their non-transformed counterparts. RESULTS: Primary feline ECs from aorta and vena cava were successfully immortalized through the successive introduction of simian virus 40 large T (SV40LT) antigen and the catalytic subunit of human telomerase (hTERT). In contrast to the parental ECs, the transformed cells were able to proliferate continuously in culture. Established cell lines exhibited several inherent endothelial properties, including typical cobblestone morphology, binding of endothelial cell-specific lectins and internalization of acetylated low-density lipoprotein. In addition, the immortalization did not affect the functional phenotype as demonstrated by their capacity to rapidly form cord-like structures on matrigel and to express cell adhesion molecules following cytokine stimulation. CONCLUSION: The ability to immortalize feline ECs, and the fact that these cells maintain the EC phenotype will enable a greater understanding of fundamental mechanisms of EC biology and endothelial-related diseases. Furthermore, the use of cell lines is an effective implementation of the 3-R principles formulated by Russel and Burch.

Heterogeneous expression of endothelial connexin (Cx) 37, Cx40, and Cx43 in rat large veins.

Gap junctions are clusters of transmembrane protein channels for intercellular communication and are composed of connexin (Cx). The vascular endothelial cells express Cx37, Cx40, and Cx43. We herein examined the spatial distribution of the endothelial connexins Cx37, Cx40, and Cx43 in rat large veins including the cranial vena cava, thoracic section of the caudal vena cava, and abdominal section of the caudal vena cava. We also examined the mean size of the endothelial cells and quantified the protein expression levels of the endothelial connexins. We found that the large veins heterogeneously expressed Cx37, Cx40, and Cx43 as follows: Cx40 > Cx37 > > Cx43 in the cranial vena cava, Cx37 > Cx43 > > Cx40 in the thoracic section of the caudal vena cava, and Cx40 > Cx43 > > Cx37 in the abdominal section of the caudal vena cava. Double immunostaining of two of the endothelial connexins revealed that the gap-junction plaques were composed of various combinations of endothelial connexins. The mean size of the endothelial cells was large, moderate, or small in the cranial vena cava, the abdominal section of the caudal vena cava, or the thoracic section of the caudal vena cava, respectively. The heterogeneity of the endothelial cells of the rat large veins in terms of the connexin expression suggests that the endothelial cells are differently coupled in the large veins. The present data are useful for investigating, for example, disease-related alterations in expression of endothelial connexins in large veins.

Selective regulation of arterial branching morphogenesis by synectin.

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.

Anatomical comparison of a defect in the hepatic segment of the inferior vena cava with the normal azygos system

During anatomical practice between 2001-2002 at the Tokyo Women's Medical University, a defect in the hepatic segment of the inferior vena cava was encountered in a 94-year-old male cadaver. Although this type of defect has often been observed clinically using imaging diagnosis, the topological changes in the small vessels and surrounding structures have not been described previously. Here, the topological changes in small vessels and surrounding structures, in addition to the morphology of a defect in the hepatic segment of the inferior vena cava, are described in detail. Normal variations in the venous system were also examined using 26 human cadavers for comparison. The following results and interpretations were obtained: The thoracic portion of the anomalous vein originated from the azygos system, judging from the topological relationships among the main venous trunk, small vessels, and the autonomic nerves. Although a close relationship between the inferior vena cava and the azygos system was recognized, the development of each venous system should be regarded as independent, based on the normal variations observed in 26 human cadavers and the currently available medical literature. The boundary between the cardinal vein (inferior vena cava) and the azygos system might be the lower border of the renal vein, since the renal vein drained into the ventral aspect of the inferior vena cava and the left renal artery ran superficially across the abdominal portion of the anomalous vein. Examination of topological changes in the surrounding structures may provide important clues regarding the morphogenesis of defects in the hepatic segment of the inferior vena cava and normal venous systems (AU)

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[Histogenesis and structural organization of the walls of rat venae cavae and pulmonary veins]. / Gistogenez i strukturnaia organizatsiia stenki polykh i legochnykh ven krysy.

Using light and electron microscopic methods, the histogenesis and structural organization of the walls of rat venae cavae and pulmonary veins were studied in prenatal and postnatal periods of development. The special attention was paid to the appearance of the striated myocytes in the walls of these vessels during the process of ontogenesis. The time of initial divergent development of myoblastic differon was established, the stages of differentiation of striated myoblasts and the peculiarities of intercellular junctions were characterized, as well as the innervation and vascularization of the walls of venae cavae and pulmonary veins.

Degradation of alpha-actin filaments in venous smooth muscle cells in response to mechanical stretch.

Mechanical stretch has been shown to induce the degradation of alpha-actin filaments in smooth muscle cells (SMC) of experimental vein grafts. Here, we investigate the possible role of ERK1/2 and p38 MAPK in regulating this process using an ex vivo venous culture model that simulates an experimental vein graft. An exposure of a vein to arterial pressure induced a significant increase in the medial circumferential strain, which induced rapid alpha-actin filament disruption, followed by degradation. The percentage of SMC alpha-actin filament coverage was reduced significantly under arterial pressure (91 +/- 1%, 43 +/- 13%, 51 +/- 5%, 28 +/- 3%, and 19 +/- 5% at 1, 6, 12, 24, and 48 h, respectively), whereas it did not change significantly in specimens under venous pressure at theses times. The degradation of SMC alpha-actin filaments paralleled an increase in the relative activity of caspase 3 (3.0 +/- 0.7- and 1.7 +/- 0.4-fold increase relative to the control level at 6 and 12 h, respectively) and a decrease in SMC density (from the control level of 1,368 +/- 66 cells/mm(2) at time 0 to 1,205 +/- 90, 783 +/- 129, 845 +/- 61, 637 +/- 55, and 432 +/- 125 cells/mm(2) at 1, 6, 12, 24, and 48 h of exposure to arterial pressure, respectively). Treatment with a p38 MAPK inhibitor (SB-203580) significantly reduced the stretch-induced activation of caspase 3 at 6 h (from 3.0 +/- 0.7- to 2.2 +/- 0.3-fold) in conjunction with a significant rescue of alpha-actin filament degradation (from 43 +/- 13% to 69 +/- 15%) at the same time. Treatment with an inhibitor for the ERK1/2 activator (PD-98059), however, did not induce a significant change in the activity of caspase 3 or the percentage of SMC alpha-actin filament coverage. These results suggest that p38 MAPK and caspase 3 may mediate stretch-dependent degradation of alpha-actin filaments in vascular SMCs.

Cellular and subcellular localization of catalase in the heart of transgenic mice.

Previous studies have described a cardiac-specific, catalase-overexpressing transgenic mouse model that was used to study myocardial oxidative injury. This study was undertaken to demonstrate cellular and subcellular localization of catalase in the hearts of transgenic mice. By the light microscopic immunoperoxidase method, we found that the overexpressed catalase was exclusively localized in cardiomyocytes. The ratios of immunoreactive cardiomyocytes in the heart were quite different among three transgenic lines examined but agreed with the elevated levels of catalase activity. In the cardiac blood vessels, positive cells were found in the walls of pulmonary veins and the vena cava, which consist of cardiomyocytes, but not in the pulmonary arteries, aorta, or cardiac valves. The electron microscopic immunogold method revealed that the elevated catalase was in sarcoplasm, nucleus, and peroxisomes, but not in mitochondria. In contrast to these distributions, catalase in the non-transgenic cardiomyocytes was in peroxisomes only. In addition, the number and size of peroxisomes in the transgenic cardiomyocytes were markedly increased, but no other ultrastructural changes were observed in comparison with those of non-transgenic mice. These results demonstrated that the elevated catalase in transgenic mouse heart is localized in cardiomyocytes and is distributed to peroxisomal and extraperoxisomal, but not mitochondrial, compartments.

Tissue factor activity is increased in human endothelial cells cultured under elevated static pressure.

We tested the hypothesis that elevated blood pressure, a known stimulus for vascular remodeling and an independent risk factor for the development of atherosclerotic disease, can modulate basal and cytokine-induced tissue factor (TF; CD 142) expression in cultured human endothelial cells (EC). Using a chromogenic enzymatic assay, we measured basal and tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml, 5 h)-induced TF activities in human aortic EC (HAEC) and vena cava EC (HVCEC) cultured at atmospheric pressure and at 170 mmHg imposed pressure for up to 48 h. Basal TF activities were 22 +/- 10 U/mg protein for HAEC and 14 +/- 9 U/mg protein for HVCEC and were upregulated in both cell types >10-fold by TNF-alpha. Exposure to pressure for 5 h induced additional elevation of basal TF activity by 47 +/- 16% (P < 0.05, n = 6) for HAEC and 17 +/- 5% (P < 0.05, n = 3) for HVCEC. Pressurization also enhanced TF activity in TNF-alpha-treated cells from 240 +/- 28 to 319 +/- 32 U/mg protein in HAEC (P < 0.05, n = 4) and from 148 +/- 25 to 179 +/- 0.8 U/mg protein (P < 0.05, n = 3) in HVCEC. Cytokine stimulation caused an approximately 100-fold increase in steady-state TF mRNA levels in HAEC, whereas pressurization did not alter either TF mRNA or cell surface antigen expression, as determined by quantitative RT-PCR methodology and ELISA. Elevated pressure, however, modulated the EC plasma membrane organization and/or permeability as inferred from the increased cellular uptake of the fluorescent amphipathic dye merocyanine 540 (33 +/- 7%, P < 0.05). Our data suggest that elevated static pressure modulates the hemostatic potential of vascular cells by modifying the molecular organization of the plasma membrane.

Human endothelial cells do not exert heparin like accelerating effects on thrombin-antithrombin-complex formation.

In the present study the formation of thrombin-antithrombin-complexes (TAT) during incubation of thrombin (0.89, 4.5, 8.9 nmol/l) and antithrombin (4.6 micromol/l) on the surface of cultured human EC, derived from different parts of the circulation, and on the surface of human vessel segments was studied. In the absence of EC TAT increased over time reaching a maximum at 60 sec; 10 sec (8.9 nmol/l thrombin): 6.35+/-0.72 nmol/l, 60 sec: 10.49+/-1.04 nmol/l. In the presence of exogenous heparin (0.1 IU/ml) maximum TAT levels were already reached after 10 sec (10.75+/-0.97); cultured EC and EC on vessel segments did not show such heparin effects. Incubation of EC with heparin resulted in an EC-surface localized heparin activity only when very high doses (3.0 IU/ml) of the drug were used. When thrombin was incubated on the EC surface in the presence of AT the efficiency of the thrombomodulin(TM)-protein C(PC)-system was markedly reduced, while in the presence of exogenous heparin (0.5 IU/l) the activity of this pathway was nearly abolished. Our results demonstrate that 1) human EC do not exert heparin-like accelerating effects on TAT formation, 2) an EC localized heparin activity is only generated when EC are incubated with amounts clearly exceeding therapeutical doses, and 3) an acceleration of TAT formation at the EC surface by heparin causes a switching off of the TM-PC-system.

Oxygen modulates alpha 1B-adrenergic receptor gene expression by arterial but not venous vascular smooth muscle.

Blood and tissue O2 levels are major determinants of short-term autoregulatory adjustments in vascular smooth muscle cell (SMC) tension and may effect long-term alterations in SMC catecholamine responsiveness. We examined the hypothesis that prolonged hypoxia altered gene expression of alpha 1-adrenoceptors. After exposure of cultured aortic (in vitro) SMC to 3% O2 for 8 h, alpha 1B mRNA increased to 523% (P = 0.02) of control cells (21% O2) and to 205% (P = 0.04) in in situ organ-cultured aortic SMC. In vivo hypoxic hypoxia (10% inspired O2) similarly increased aortic SMC alpha 1B mRNA 180% (P = 0.02). In contrast, alpha 1D, alpha-actin and beta-actin mRNA levels were not changed in aortic SMC by low O2 in the in vitro, in situ, or in vivo models. Unlike aortic SMC, vena caval SMC alpha 1B mRNA expression did not change with low-O2 exposure in vitro or in vivo, nor did alpha 1D, alpha-actin or beta-actin mRNA. Aortic SMC alpha 1B transcription rate increased 360% (P = 0.02), whereas alpha 1D, alpha-actin, and beta-actin transcription was unchanged. Neither alpha 1B nor alpha 1D mRNA stability was altered by low-O2 exposure. Total alpha 1-adrenoceptor density ([3H]prazosin binding) increased 12% (P = 0.04) after 24 h of 3% O2. This was associated with a 200% increase (P < 0.01) in the chloroethylclonidine (CEC)-sensitive alpha 1-adrenoceptor population and no change in CEC-insensitive alpha 1-adrenoceptor density. Exposure of aortic SMC to 24 h of 3% O2 increased the maximum response of norepinephrine-evoked elevations in intracellular Ca2+ as measured using fura 2. Low O2 did not change responses to another G protein-coupled receptor, angiotensin II. These data suggest that reduced O2, during prolonged hypoxemia or tissue ischemia, may selectively increase expression of functionally coupled alpha 1B-adrenoceptors in arterial blood vessels.

Different temporal and spatial distribution of TGF-beta 1 and TGF-beta 2 in rabbit vascular tissue: potential role in normal vessel growth and maturation.

The expression of transforming growth factor beta 1 and beta 2 (TGF-beta 1 and beta 2) in aortic and venous tissue from male New Zealand White rabbits, at selected time intervals after birth, was examined by the reverse transcriptase polymerase chain reaction. Stable levels of TGF-beta 1 were found in all segments derived from the aortic arch and descending aorta at each time interval. However, increasing amounts of TGF-beta 2 transcripts were observed for the aortic arch from day 4, with peaks occurring between 1 and 6 months of age, followed by progressively decreasing levels thereafter. TGF-beta 2 transcripts in the descending aorta generally did not change significantly over time. TGF-beta transcripts manifested a significantly lower expression in the vena cava than in aortic segments. Histological analysis of the vascular tissue showed cellular hyperplasia (2.5-fold greater prevalence of nuclei per field) in the aortic arch media at 1 month of age as compared with nuclei per field at 12 months and increasing thickness of the aortic arch media with time. No significant differences in relative collagen concentrations were observed among the aortic and vena cava segments. These results suggest that these TGF-beta isoforms may participate in the physiological induction and differentiation of arterial and venous tissue during early normal vascular maturation.

Development of cardiac musculature in the cranial vena cava of rat embryos.

Development of cardiac musculature in the rat cranial vena cava (common cardinal vein or duct of Cuvier) was examined by immunohistochemistry and transmission electron microscopy. Undifferentiated cardiac myocytes were detected in the cranial vena cava wall of rat embryos after 12.5 days post-coitum (dpc). The tunica media of the cranial vena cava was composed of cardiac myocytes after formation of the endothelium. Therefore, the cranial vena cava may be not only a part of the venous system but also of the heart. Myocytes in the cranial vena cava contained developing myofibrils, mitochondria and intercalated discs similar to those found in the myocytes in heart. Striated myofibrils began to differentiate as soon as myocytes appeared in the vena cava wall, and myocytes with differentiating myofibrils occur in the wall as the first component of the tunica media at 12.5 dpc. We concluded that the cardiac musculature in the vena cava is not a secondary extension into the tunica media after birth only in the rat, but a basic structure formed in all mammals during early embryonic development.

Regulation of vascular smooth muscle growth by alpha 1-adrenoreceptor subtypes in vitro and in situ.

Rat aorta smooth muscle cells which express all three alpha 1-adrenoreceptors (alpha 1A, alpha 1B and alpha 1D) were used to determine the effect of stimulation of alpha 1-adrenergic receptor subtypes on cell growth. "Combined" alpha 1-adrenoreceptor subtype stimulation with norepinephrine alone caused a concentration-dependent, prazosin-sensitive increase in protein content and synthesis: 48 h of stimulation at 1 microM increased cell protein to 216 +/- 40% of time-matched controls (p = 0.008) and RNA to 140 +/- 13% (p = 0.03); protein synthesis increased to 167 +/- 13% (p < 0.01) after 24 h. Stimulation with norepinephrine plus the selective alpha 1A/alpha 1D antagonist 5-methylurapidil produced greater increases in alpha-actin mRNA (270 +/- 40% at 8 h; p = 0.007), total cell protein (220 +/- 45% at 24 h; p = 0.004), and RNA (135 +/- 8% at 24 h; p = 0.01). These effects were prevented by pretreatment with the selective alpha 1B antagonist chloroethylclonidine. Comparable results were obtained for intact aortae. Stimulation with norepinephrine plus 5-methylurapidil increased (p < 0.05) tissue protein, RNA, dry weight, and alpha-actin mRNA; and as in culture cells, combined stimulation with norepinephrine alone attenuated these responses. By comparison, adventitia (fibroblasts) was unaffected. Removal of endothelial cells had no effect. alpha 1B mRNA decreased by 42 +/- 12% (p = 0.01) in cultured cells during combined alpha 1-adrenoreceptor stimulation and by 23 +/- 8% (p = 0.03) for intact aorta. alpha 1D and beta-actin mRNA were unchanged in cultured cells, aorta media, and adventitia. These findings suggest that prolonged stimulation of chloroethylclonidine-sensitive, possibly alpha 1B-adrenoceptors induces hypertrophy of arterial smooth muscle cells and that stimulation of 5-methylurapidil-sensitive, non-alpha 1B-adrenoreceptors attenuates this growth response.

Cardiac musculature of the cranial vena cava in the common tree shrew (Tupaia glis).

Cardiac musculature of the cranial vena cava in the common tree shrew (Tupaia glis) was examined by light and transmission electron microscopy. The common tree shrew has well developed cardiac myocyte layers in the tunica media of the cranial vena cava, extending from the right atrium to the root of the subclavian vein. Because the common tree shrew belongs to a primitive group of mammals, the occurrence of cardiac musculature in the cranial vena cava may be a common feature in lower mammals. The development of this musculature indicates that active contraction of the cranial vena cava wall occurs in this species. Electron micrographs showed the typical ultrastructure of myocytes and nerve endings. These observations suggest that this musculature may serve as a regulatory pump for the return of venous blood to the right atrium and as a blood reservoir system under conditions of rapid heart rate. Additionally, the presence of atrial natriuretic polypeptide (ANP) was also demonstrated in the myocytes of the vena cava immunohistochemically. These findings show that the cardiac endocrine organ for ANP develops even in the principal veins including the cranial vena cava.

Physiologic responses of REVC, a continuous rabbit endothelial vascular cell.

Endothelial cells (EC) are fastidious in their growth requirements in vitro and will not survive extensive passages. We have partially characterized a continuous cell line (> 40 passages) established in culture from New Zealand White rabbit vena cava endothelium (REVC). REVC cells resemble typical EC, but remain hardy when grown on uncoated plastic in DMEM/F12 + 10% FBS. REVC cells have typical cobblestone appearance, are contact-inhibited in monolayers and express factor VIII-related antigen. Weibel-Palade bodies were not seen by electron microscopy. REVC cells grown in 2% FBS on plastic demonstrate dose-dependent increases in [3H]thymidine uptake in response to acidic FGF (10-100 ng/ml), basic FGF (3-100 ng/ml), EGF (10-50 ng/ml), and ECGS (10-100 micrograms/ml). Heparin (5-100 micrograms/ml) potentiates proliferation induced by aFGF and lowered the ED50 for aFGF. REVC cells did not show an increased proliferative rate in response to vascular endothelial growth factor. Transforming growth factor beta 1 and beta 2 profoundly inhibited thymidine uptake at doses as low as 100 pg/ml. When grown on a collagen I substratum, REVC cells became larger, more polygonal and assumed a sheet-like appearance upon reaching confluence. REVC cells plated on fibronectin, laminin or poly-L-lysine demonstrated increases in pericellular granularity and pronounced spreading, especially on fibronectin. Phorbol myristate acetate produced profound morphological changes characterized by swirling whorls of bipolar cells surrounding patches of polygonal cells and multilayered overgrowth. When plated on EHS (Engelbreth-Holm-Swarm) tumor extracellular matrix (Matrigel), REVC cells became quiescent and underwent morphological changes reminiscent of differentiation with elongated cytoplasmic extensions. Chromosomal examination of REVC cells revealed a normal diploid karyotype (2n = 44). This continuous cell line is undergoing further characterization and may be quite useful in investigating many aspects of endothelial cell biology in vitro.

Contributions of K+, Na+, and Cl- to the membrane potential of intact hamster vascular endothelial cells.

The transmembrane potential (Vm) of vascular endothelial cells (EC) is an important property that may be involved in intra- and intercellular signal transduction for various vascular functions. In this study, Vm of intact aortic and vena caval EC from hamsters were measured using conventional microelectrodes. Vascular strips with the luminal surface upwards were suffused in a tissue chamber with Krebs solution in physiological conditions. The resting Vm of aortic and vena caval EC was found to be -40 +/- 1 mV (n = 55) and -43 +/- 1 mV (n = 15), respectively. The Vm recordings were confirmed to have originated from EC by scanning and transmission electron microscopy combined with the comparison of electrical recordings between normal and endothelium-denuded aortic strips. The input resistance varied from 10-240 M omega, which implied the presence of electrical coupling between vascular EC. Elevating the K+ level in the suffusate from 4.7 mM to 50 and 100 mM depolarized aortic EC by 19% and 29% and vena caval EC by 18% and 29%, respectively. These low percentages indicated a relatively small contribution of [K+] to the resting Vm of vascular EC. A positive correlation (r > 0.69) between the resting Vm and the magnitude of depolarization by the high [K+]o may be related to the involvement of voltage-dependent K+ channels. The hyperpolarization caused by lowering both [Na+]o and [Cl-]o suggested the disengagement of some electrogenic transport systems in the membrane, such as a Na(+)-K(+)-Cl- cotransporter. The transference number (t(ion)), as an index of membrane conductance for specific ions, was calculated for K+ (15-20%), Na+ (16%), and Cl- (9-15%), demonstrating that both Na+ and Cl- as well as K+ contribute to the overall resting Vm. Our study documented some basic electrophysiology of the vascular EC when both structural and functional properties of the cell were maintained, thus furthering the understanding of the essential role of endothelial cells in mediating vascular functions.

Effect of dihydropyridines on calcium channels in isolated smooth muscle cells from rat vena cava.

1. Whole-cell patch-clamp method was applied to single smooth muscle cells freshly isolated from the rat inferior vena cava. 2. Depolarizing pulses, applied from a holding potential of -90 mV, activated both Na+ and Ca2+ channels. The fast Na+ current was inhibited by nanomolar concentrations of tetrodotoxin (TTX). The slow Ba2+ current (measured in 5 mM Ba2+ solution) was inhibited by Cd2+ and modulated by dihydropyridine derivatives. When the cells were held at a holding potential of -80 mV, racemic Bay K 8644 increased the Ba2+ current (ED50 = 10 nM) while racemic isradipine inhibited the current (IC50 = 21 nM). 3. The voltage-dependency of isradipine blockade was assessed by determining the steady-state availability of the Ca2+ channels. From the shift of the inactivation curve in the presence of isradipine, we calculated a dissociation constant of 1.11 nM for inactivated Ca2+ channels. Scatchard plots of the specific binding of (+)-[3H]-isradipine obtained in intact strips incubated in 5.6 mM or 135 mM K+ solutions confirmed the voltage-dependency of isradipine binding. 4. Specific binding of (+)-[3H]-isradipine was completely displaced by unlabelled (+/-)-isradipine, with an IC50 of 15.1 nM. This value is similar to the IC50 for inhibition of the Ba2+ current (21 nM) in cells maintained at a holding potential of -80 mV. 5. Bay K 8644 had no effects on the Ba2+ current kinetics during a depolarizing test pulse. The steady-state inactivation-activation curves of Ba2+ current were not significantly shifted along the voltage axis.6. The present data suggest the existence of two distinct dihydropyridine binding sites which can be bound preferentially by agonist or antagonist derivatives.

Atherosclerosis- and age-related multinucleated variant endothelial cells in primary culture from human aorta.

Endothelial cells were cultured from human aortas and inferior venae cavae of autopsied subjects ranging in age from infancy to 85 years. Endothelial cells in 32 of more than 100 attempted cultures were pure enough for evaluation. Emerged endothelial cells in primary culture were classified into two types: typical endothelium and variant endothelium. Typical endothelial cells were small, round to polygonal shaped, and were arranged uniformly. Their diameter ranged from 50 to 70 microns. Variant endothelial cells were larger, ranging from 100 to 200 microns in diameter, and giant endothelial cells measuring more than 250 microns in diameter were scattered among them. Variant endothelial cells were usually multinucleated and possessed endothelium-specific markers of vWF and Weibel-Palade bodies. No incorporation of [3H]thymidine was found in the nuclei of cultured variant endothelial cells. Although most cultured endothelial cells were of the typical type, variant endothelial cells were interspersed throughout the culture. The ratio of variant endothelial cells to typical cells correlated well with the severity of atherosclerosis, but less so with aging. The number of variant endothelial cells in cultures from inferior venae cavae was slight and constant throughout all age groups. The presence of multinucleated endothelial cells in in vivo aortas was confirmed by both scanning and transmission electron microscopy. They sometimes existed in colonies in the aortas from elderly subjects with intimal-thickened or advanced atherosclerotic lesions. These results indicate that variant endothelial cells were present in vivo and their ratio in primary culture reflected the in vivo population. It is likely that these cells were formed by adhesion of adjacent typical endothelial cells and that this process was affected more by atherosclerosis than by aging. Although it is not clear if the multinucleated variant cells were formed before the formation of atherosclerotic plaque or after the plaque formation, they will contribute to further development of atherosclerotic lesions, which in turn cause malfunction of the cell membrane. We suggest that there is a cyclic effect of these processes for multiplication of the variant endothelial cells and advancement of atherosclerotic lesions.

A simple, practical 'swiss roll' method of preparing tissues for paraffin or methacrylate embedding.

A simple method of preparing 'swiss rolls' from strips of tissue up to 500 mm in length is described. This procedure has been applied to a variety of tissues and is suitable for the preparation of both paraffin- and methacrylate-embedded specimens.