Recruitment of smooth muscle cells and arterial vasomotion

Investigating the recruitment and synchronization of smooth muscle cells (SMCs) is the key to understanding the physical mechanisms leading to contraction and spontaneous diameter oscillations of arteries, called vasomotion. We improved a method that allows the correlation of calcium oscillations (flashing) of individual SMCs with mean calcium variations and arterial contraction using confocal microscopy. Endothelium-stripped rat mesenteric arteries were cut open, loaded with dual calcium fluorescence probes, and stimulated by increasing concentrations of the vasoconstrictors phenylephrine (PE) and KCl. We found that the number and synchronization of flashing cells depends on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells flash asynchronously and no local contraction is detected. At medium concentration, recruitment of cells is complete and synchronous, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous calcium oscillations and fully contracted vessels, whereas high concentration of KCl leads to a sustained nonoscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling rat mesenteric artery contraction and vasomotion.     

Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40

Control of cerebral vasculature differs from that of systemic vessels outside the blood-brain barrier. The hypothesis that the endothelium modulates vasomotion via direct myoendothelial coupling was investigated in a small vessel of the cerebral circulation. In the primary branch of the rat basilar artery, membrane potential, diameter, and calcium dynamics associated with vasomotion were examined using selective inhibitors of endothelial function in intact and endothelium-denuded arteries. Vessel anatomy, protein, and mRNA expression were studied using conventional electron microscopy high-resolution ultrastructural and confocal immunohistochemistry and quantitative PCR. Membrane potential oscillations were present in both endothelial cells and smooth muscle cells (SMCs), and these preceded rhythmical contractions during which adjacent SMC intracellular calcium concentration ([Ca(2+)](i)) waves were synchronized. Endothelium removal abolished vasomotion and desynchronized adjacent smooth muscle cell [Ca(2+)](i) waves. N(G)-nitro-l-arginine methyl ester (10 microM) did not mimic this effect, and dibutyryl cGMP (300 muM) failed to resynchronize [Ca(2+)](i) waves in endothelium-denuded arteries. Combined charybdotoxin and apamin abolished vasomotion and depolarized and constricted vessels, even in absence of endothelium. Separately, (37,43)Gap27 and (40)Gap27 abolished vasomotion. Extensive myoendothelial gap junctions (3 per endothelial cell) composed of connexins 37 and 40 connected the endothelial cell and SMC layers. Synchronized vasomotion in rat basilar artery is endothelium dependent, with [Ca(2+)](i) waves generated within SMCs being coordinated by electrical coupling via myoendothelial gap junctions.     

Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger

We investigated heterocellular communication in rat mesenteric arterial strips at the cellular level using confocal microscopy. To visualize Ca(2+) changes in different cell populations, smooth muscle cells (SMCs) were loaded with Fluo-4 and endothelial cells (ECs) with Fura red. SMC contraction was stimulated using high K(+) solution and Phenylephrine. Depending on vasoconstrictor concentration, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in a subpopulation of ECs 5-11s after a [Ca(2+)](i) rise was observed in adjacent SMCs. This time interval suggests chemical coupling between SMCs and ECs via gap junctions. As potential chemical mediators we investigated Ca(2+) or inositol 1,4,5-trisphosphate (IP(3)). First, phospholipase C inhibitor U-73122 was added to prevent IP(3) production in response to the [Ca(2+)](i) increase in SMCs. In high K(+) solution, all SMCs presented global and synchronous [Ca(2+)](i) increase, but no [Ca(2+)](i) variations were detected in ECs. Second, 2-aminoethoxydiphenylborate, an inhibitor of IP(3)-induced Ca(2+) release, reduced the number of flashing ECs by 75+/-3% (n = 6). The number of flashing ECs was similarly reduced by adding the gap junction uncoupler palmitoleic acid. Thus, our results suggest a heterocellular communication through gap junctions from SMCs to ECs by diffusion, probably of IP(3).     

Role of the endothelium on arterial vasomotion

It is well-known that cyclic variations of the vascular diameter, a phenomenon called vasomotion, are induced by synchronous calcium oscillations of smooth muscle cells (SMCs). However, the role of the endothelium on vasomotion is unclear. Some experimental studies claim that the endothelium is necessary for synchronization and vasomotion, whereas others report rhythmic contractions in the absence of an intact endothelium. Moreover, endothelium-derived factors have been shown to abolish vasomotion by desynchronizing the calcium signals in SMCs. By modeling the calcium dynamics of a population of SMCs coupled to a population of endothelial cells, we analyze the effects of an SMC vasoconstrictor stimulation on endothelial cells and the feedback of endothelium-derived factors. Our results show that the endothelium essentially decreases the SMCs calcium level and may move the SMCs from a steady state to an oscillatory domain, and vice versa. In the oscillatory domain, a population of coupled SMCs exhibits synchronous calcium oscillations. Outside the oscillatory domain, the coupled SMCs present only irregular calcium flashings arising from noise modeling stochastic opening of channels. Our findings provide explanations for the published contradictory experimental observations.     

Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition

In resistance arteries, endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). Endothelial nitric oxide synthase (eNOS) is present in the luminal side of the EC (apical EC) and the basal side of the EC (MEJ). To test if these eNOS pools acted in sync or separately, we co-cultured ECs and SMCs, then stimulated SMCs with phenylephrine (PE). Adrenergic activation causes inositol [1,4,5] triphosphate (IP₃) to move from SMC to EC through gap junctions at the MEJ. PE increases MEJ eNOS phosphorylation (eNOS-P) at S1177, but not in EC. Conversely, we used bradykinin (BK) to increase EC calcium; this increased EC eNOS-P but did not affect MEJ eNOS-P. Inhibiting gap junctions abrogated the MEJ eNOS-P after PE, but had no effect on BK eNOS-P. Differential lipid composition between apical EC and MEJ may account for the compartmentalized eNOS-P response. Indeed, DAG and phosphatidylserine are both enriched in MEJ. These lipids are cofactors for PKC activity, which was significantly increased at the MEJ after PE. Because PKC activity also relies on endoplasmic reticulum (ER) calcium release, we used thapsigargin and xestospongin C, BAPTA, and PKC inhibitors, which caused significant decreases in MEJ eNOS-P after PE. Functionally, BK inhibited leukocyte adhesion and PE caused an increase in SMC cGMP. We hypothesize that local lipid composition of the MEJ primes PKC and eNOS-P for stimulation by PE, allowing for compartmentalized function of eNOS in the blood vessel wall.     

Mechanism of constrictive vascular remodeling by homocysteine: role of PPAR

To test the hypothesis that homocysteine induces constrictive vascular remodeling by inactivating peroxisome proliferator-activated receptor (PPAR), aortic endothelial cells (ECs) and smooth muscle cells (SMCs) were isolated. Collagen gels were prepared, and ECs or SMCs (10(5)) or SMCs + ECs (10(4)) were incorporated into the gels. To characterize PPAR, agonists of PPAR-alpha [ciprofibrate (CF)] and PPAR-gamma [15-deoxy-12,14-prostaglandin J(2) (PGJ(2))] were used. To determine the role of disintegrin metalloproteinase (DMP), cardiac inhibitor of metalloproteinase (CIMP) was used in collagen gels. Gel diameter at 0 h was 14.1 +/- 0.2 mm and was unchanged up to 24 h as measured by a digital micrometer. SMCs reduce gel diameter to 10.5 +/- 0.4 mm at 24 h. Addition of homocysteine to SMCs reduces further the gel diameter to 8.0 +/- 0.2 mm, suggesting that SMCs induce contraction and that the contraction is further enhanced by homocysteine. Addition of ECs and SMCs reduces gel diameter to 12.0 +/- 0.3 mm, suggesting that ECs play a role in collagen contraction. Only PGJ(2), not CF, inhibits SMC contraction. However, both PGJ(2) and CF inhibit contraction of ECs and SMCs + ECs. Addition of anti-DMP blocks SMC- as well as homocysteine-mediated contraction. However, CIMP inhibits only homocysteine-mediated contraction. The results suggest that homocysteine may enhance vascular constrictive remodeling by inactivating PPAR-alpha and -gamma in ECs and PPAR-gamma in SMCs.     

Role of membrane potential in vasomotion of isolated pressurized rat arteries

Vasomotion, the phenomenon of vessel diameter oscillation, regulates blood flow and resistance. The main parameters implicated in vasomotion are particularly the membrane potential and the cytosolic free calcium in smooth muscle cells. In this study, these parameters were measured in rat perfused-pressurized mesenteric artery segments. The application of norepinephrine (NE) caused rhythmic diameter contractions and membrane potential oscillations (amplitude; 5.3 +/- 0.3 mV, frequency; 0.09 +/- 0.01 Hz). Verapamil (1 microM) abolished this vasomotion. During vasomotion, 10(-5) M ouabain (Na(+)-K(+) ATPase inhibitor) decreased the amplitude of the electrical oscillations but not their frequency (amplitude; 3.7 +/- 0.3 mV, frequency; 0.08 +/- 0.002 Hz). Although a high concentration of ouabain (10(-3) M) (which exhibits non-specific effects) abolished both electrical membrane potential oscillations and vasomotion, we conclude that the Na+-K+ ATPase could not be implicated in the generation of the membrane potential oscillations. We conclude that in rat perfused-pressurized mesenteric artery, the slow wave membrane type of potential oscillation by rhythmically gating voltage-dependent calcium channels, is responsible for the oscillation of intracellular calcium and thus vasomotion.     

Effects of the lysosomal fraction of polymorphonuclear leukocytes on proliferation of cultured vascular cells

The effects of the lysosomal fraction isolated from polymorphonuclear leukocytes (PLF) on the growth of cultivated aortic medial smooth muscle cells (SMCs) and arterial endothelial cells (ECs) were studied by assaying DNA synthesis and counting the numbers of cells. PLF proved to promote the growth of cultivated SMCs and ECs. There was a positive correlation between an increase in DNA synthesis and the dose of PLF. The growth-promoting effect was observed in sparsely cultivated SMCs and ECs, in densely cultivated SMCs, but not in confluently cultivated ECs. The difference in response between SMCs and ECs seems to depend on their biological characteristics. Because a small amount of PLF showed potent growth-promoting activity in the presence of 10% fetal bovine serum which possesses a high protease blocking activity, the mechanism of this promoting activity is suggested to be independent of the proteases contained in PLF.     

Improved arterial wall model by coculturing vascular endothelial and smooth muscle cells

We have constructed an in vitro arterial wall model by coculturing bovine arterial endothelial cells (ECs) and smooth muscle cells (SMCs). When ECs were seeded directly over SMCs and cocultured in an ordinary culture medium, ECs grew sparsely and did not form a confluent monolayer. Addition of ascorbic acid to the culture medium at concentrations greater than 50 μg/ml increased the production of type IV collagen by the SMCs, and ECs formed a confluent monolayer covering the entire surface of SMCs. Histological studies showed that the thickness of the cell layer composed of ECs and SMCs increased with increasing duration of coculture. This arterial wall model, prepared by our method, may serve as a simple and good in vitro model to study the effects of factors such as biological chemicals and shear stress on cell proliferation and other physiological functions of arterial walls.     

The effect of coculture with human smooth muscle cells on the proliferation,the IL‐1 β secretion,the PDGF production and tube formation of human aortic endothelial cells

Endothelial cells (ECs) and smooth muscle cells (SMCs), which are the major component cells of blood vessels, produce various bioactive substances and communicate with each other through them. Although several studies of the interaction between ECs and SMCs have been reported, the effect of coculture with SMCs on ECs is still obscure. To clarify the interaction of ECs and SMCs, we examined the effect of coculture with SMCs on the proliferation, the IL‐1β secretion, the PDGF production and tube formation of ECs, using the coculture model: transferable wells and collagen gel. IL‐1 and PDGF are considered to be related to progression of atherosclerosis. Proliferation and tube formation of ECs are associated with repair of vessels. In the transferable well system coculture with SMCs stimulated the proliferation of ECs, and enhanced the IL‐1β secretion of ECs and in the collagen gel system coculture with SMCs induced the tube formation of ECs, and appeared to enhance the PDGF production of ECs. In conclusion, the effect of coculture with SMCs on ECs has two conflicting aspects: progression of atherosclerosis and angiogenesis. These results suggest that an imbalance of their effects may lead to pathological events. Copyright © 1999 John Wiley & Sons, Ltd.     

Effects of arterial wall stress on vasomotion

Smooth muscle and endothelial cells in the arterial wall are exposed to mechanical stress. Indeed blood flow induces intraluminal pressure variations and shear stress. An increase in pressure may induce a vessel contraction, a phenomenon known as the myogenic response. Many muscular vessels present vasomotion, i.e., rhythmic diameter oscillations caused by synchronous cytosolic calcium oscillations of the smooth muscle cells. Vasomotion has been shown to be modulated by pressure changes. To get a better understanding of the effect of stress and in particular pressure on vasomotion, we propose a model of a blood vessel describing the calcium dynamics in a coupled population of smooth muscle cells and endothelial cells and the consequent vessel diameter variations. We show that a rise in pressure increases the calcium concentration. This may either induce or abolish vasomotion, or increase its frequency depending on the initial conditions. In our model the myogenic response is less pronounced for large arteries than for small arteries and occurs at higher values of pressure if the wall thickness is increased. Our results are in agreement with experimental observations concerning a broad range of vessels.     

A model of smooth muscle cell synchronization in the arterial wall

Vasomotion is a rhythmic variation in microvascular diameter. Although known for more than 150 years, the cellular processes underlying the initiation of vasomotion are not fully understood. In the present study a model of a single cell is extended by coupling a number of cells into a tube. The simulated results point to a permissive role of cGMP in establishing intercellular synchronization. In sufficient concentration, cGMP may activate a cGMP-sensitive calcium-dependent chloride channel, causing a tight spatiotemporal coupling between release of sarcoplasmic reticulum calcium, membrane depolarization, and influx of extracellular calcium. Low [cGMP] is associated only with unsynchronized waves. At intermediate concentrations, cells display either waves or whole cell oscillations, but these remain unsynchronized between cells. Whole cell oscillations are associated with rhythmic variation in membrane potential and flow of current through gap junctions. The amplitude of these oscillations in potential grows with increasing [cGMP], and, past a certain threshold, they become strong enough to entrain all cells in the vascular wall, thereby initiating sustained vasomotion. In this state there is a rhythmic flow of calcium through voltage-sensitive calcium channels into the cytoplasm, making the frequency of established vasomotion sensitive to membrane potential. It is concluded that electrical coupling through gap junctions is likely to be responsible for the rapid synchronization across a large number of cells. Gap-junctional current between cells is due to the appearance of oscillations in the membrane potential that again depends on the entrainment of sarcoplasmic reticulum and plasma membrane within the individual cell.     

Analysis of effects of connexin-mimetic peptides in rat mesenteric small arteries

Synthetic peptides homologous to the extracellular loops of the major vascular connexins represent a novel class of gap junction blockers that have been used to assess the role of direct cellular communication in arteries and veins. However, the specificity of action of such peptides on the coupling between smooth muscle cells (SMCs) has not yet been fully characterized. Isolated third-order rat mesenteric arteries were therefore studied with respect to isometric tension (myography), intracellular Ca2+ concentration ([Ca2+]i) (Ca2+ -sensitive dyes), membrane potential, and input resistance (sharp intracellular glass electrodes). Confocal imaging was used for visualization of [Ca2+]i events in individual SMCs in the arterial wall and membrane currents (patch clamp) measured in individual SMCs isolated from the same arteries. A triple peptide combination (37,43Gap 27 + 40Gap 27 + 43Gap 26) increased intercellular resistance (measured as input resistance) in intact arterial segments without affecting the membrane conductance of individual cells and also interrupted electrical coupling between pairs of rat aortic A7r5 myocytes. In intact arterial segments, the peptides desynchronized [Ca2+]i transients in individual SMCs and abolished vasomotion without suppressing Ca2+ transients in individual cells. They also depolarized SMCs, increased [Ca2+]i, and attenuated acetylcholine-induced, endothelium-dependent smooth muscle hyperpolarization. Experiments with endothelium-denuded arteries suggested that the depolarization produced by the peptides under basal conditions was in part secondary to electrical uncoupling of the endothelium from SMCs with loss of a tonic hyperpolarizing effect of the endothelium. Taken together, the results indicate that connexin-mimetic peptides block electrical signaling in rat mesenteric small arteries without exerting major nonjunctional effects.     

A model for studying the effect of shear stress on interactions between vascular endothelial cells and smooth muscle cells

Vascular endothelial cells (ECs) are constantly subjected to blood flow-induced shear stress and the influences of neighboring smooth muscle cells (SMCs). In the present study, a coculture flow system was developed to study the effect of shear stress on EC-SMC interactions. ECs and SMCs were separated by a porous membrane with only the EC side subjected to the flow condition. When ECs were exposed to a shear stress of 12 dynes/cm2 for 24 h, the cocultured SMCs tended to orient perpendicularly to the flow direction. This perpendicular orientation of the cocultured SMCs to flow direction was not observed when ECs were exposed to a shear stress of 2 dynes/cm2. Under the static condition, long and parallel actin bundles were observed in the central regions of the cocultured SMCs, whereas the actin filaments localized mainly at the periphery of the cocultured ECs. After 24 h of flow application, the cocultured ECs displayed very long, well-organized, parallel actin stress fibers aligned with the flow direction in the central regions of the cells. Immunostaining of platelet endothelial cell adhesion molecule-1 confirmed the elongation and alignment of the cocultured ECs with the flow direction. Coculture with SMCs under static condition induced EC gene expressions of growth-related oncogene-alpha and monocyte chemotactic protein-1, and shear stress was found to abolish these SMC-induced gene expressions. Our results suggest that shear stress may serve as a down-regulator for the pathophysiologically relevant gene expression in ECs cocultured with SMCs.     

内皮细胞生长状态对血管平滑肌细胞增生迁移的影响

实验通过建立细胞共培养体系,探讨内皮细胞生长状态对血管平滑肌细胞增生迁移的影响及机制。检测指标包括~3H-TdR掺入、细胞周期、细胞迁移计数和α-SM-actin mRNA表达。结果显示,融合生长内皮使平滑肌细胞~3H-TdR掺入量明显降低,增加平滑肌细胞停留在G_0/G_1期的比例,上调平滑肌细胞α-SM-actin mRNA表达;而对数生长内皮细胞使平滑肌细胞~3H-TdR掺入量明显升高,促进平滑肌细胞由 G_0/G_1期进入G_2/M和S期,下调平滑肌细胞α-SM-actin mRNA表达。对照组平滑肌细胞在基础状态下存在少量迁移,对数增殖内皮细胞组平滑肌迁移数比对照组增高约4倍(P<0.01),而融合生长内皮细胞组平滑肌迁移数仅为对照组的0.5倍(P<0.05)。结果提示内皮细胞生长状态不同,对平滑肌细胞生物学特性的影响也不同,增殖期内皮明显促进平滑肌细胞增生迁移、下调平滑肌细胞α-SM-actin mRNA表达。     

The nonstructural glycoprotein of rotavirus affects intracellular calcium levels.

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirus-infected cells.     

26-hydroxycholesterol-stimulated DNA synthesis in smooth muscle cells and induction of endothelial injury using a coculture technique

We investigated the effects of 0.5, 2.5, and 10 micrograms/ml of cholesterol or 26-hydroxycholesterol on bovine aortic ECs and SMCs. Suppression of viable cell density and cytotoxic changes in both cells were induced by 2.5 and 10 micrograms/ml 26-hydroxycholesterol. ECs were more severely damaged than SMCs in the presence of 26-hydroxycholesterol. Levels of up to 10 micrograms/ml cholesterol had no effect on ECs or SMCs growth or cytotoxicity. Confluent ECs exposed to 2.5 or 10 micrograms/ml of 26-hydroxycholesterol secreted significant amounts of 6-ketoprostaglandin F1 alpha after a 24-hr incubation. An equivalent concentration of cholesterol had no such effect. SMCs cocultured with ECs exposed to 10 micrograms/ml 26-hydroxycholesterol, when compared with an equivalent level of cholesterol, synthesized DNA in significantly greater amounts during a 24-hr incubation. The cocultured ECs incubated in the presence of 2.5 and 10 micrograms/ml 26-hydroxycholesterol were partially detached due to cell death. However, no difference was observed in the DNA content of SMCs cultured without ECs in the presence of cholesterol or 26-hydroxycholesterol. The results suggest that 26-hydroxycholesterol produced not only cytotoxicity to ECs and SMCs but also stimulated DNA synthesis in SMCs through endothelial injury.     

Simultaneous isolation of endothelial and smooth muscle cells from human umbilical artery or vein and their growth response to low-density lipoproteins

In the pathogenesis of atherosclerosis the interplay of endothelial cells (ECs) and smooth muscle cells (SMCs) is disturbed. Oxidatively modified low-density lipoproteins (oxLDLs), important stimulators of atherosclerotic plaque formation in vessels, modify the growth response of both cell types. To compare growth responses of ECs and SMCs of the same vessel with oxLDLs, we developed a method to isolate both cell types from the vessel walls of umbilical cords by enzymatic digestion. The method further allowed the simultaneous isolation of venous and arterial cells from a single umbilical cord. In culture, venous ECs showed an elongated appearance compared with arterial ECs, whereas SMCs of artery and vein did not look different. Smooth muscle cells of both vessel types responded to oxLDLs (60 microg/ml) with an increase in their [(3)H]-thymidine incorporation into DNA. On the contrary, ECs of artery or vein decreased [(3)H]-thymidine incorporation and cell number in the presence of oxLDLs (60 microg/ml) of increasing oxidation grade. Thus, human umbilical SMCs and ECs of the same vessel show a disparate growth response toward oxLDLs. But the physiologically more relevant minimal oxLDLs did not decrease proliferation in venous ECs but only in arterial ECs. This difference in tolerance toward minimal oxLDLs should be taken into account while using venous or arterial ECs of umbilical cord for research in atherosclerosis. Further differences of venous and arterial ECs in tolerance toward minimal oxLDLs could be of clinical relevance for coronary artery bypass grafts.     

Pulmonary arterial smooth muscle cells modulate cytokine- and LPS-induced cytotoxicity in endothelial cells

Cytokines and lipopolysaccharide (LPS) are known to be injurious to vascular endothelial cells (ECs), but the influence of adjacent vascular smooth muscle cells (SMCs) on this injury is unknown. Exposure of cultured rat (RPMECs) or human (HPMECs) pulmonary microvascular ECs on tissue culture plastic to a mixture of cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) and LPS (cytomix) resulted in a significant increase in (51)Cr release to 35-40%. When unstimulated RPMECs were cocultured with cytomix-pretreated rat pulmonary microvascular SMCs (RPMSMCs) there was an increase in (51)Cr release to 8.4%, which was nitric oxide dependent. However, when RPMECs or HPMECs were stimulated in direct contact with their respective SMCs, rather than a further increase in cytomix-induced injury (e.g., >35-40%), (51)Cr release decreased to <10%. This cytoprotection was fully reproduced with fixed RPMSMCs, and partially reproduced by plating HPMECs on gelatin. These data show that the direct toxicity of a cytokine and endotoxin mixture on cultured ECs can be reduced by contact with vascular smooth muscle.     

Folding kinetics of the lipoic acid-bearing domain of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex

Intracellular calcium is a second messenger involved in several processes in yeast, such as mating, nutrient sensing, stress response and cell cycle events. It was reported that glucose addition stimulates a rapid increase in free calcium level in yeast. To investigate the calcium level variations induced by different stimuli we used a reporter system based on the photoprotein aequorin. Glucose addition (50 mM) to nutrient-starved cells induced an increase in free intracellular calcium concentration, mainly due to an influx from external medium. The increase of calcium reached its maximum 100–120 s after the stimulus. A concentration of about 20 mM glucose was required for a 50% increase in intracellular calcium. This response was completely abolished in strain plc1Δ and in the isogenic wild-type strain treated with 3-nitrocoumarin, a phosphatidylinositol-specific phospholipase C inhibitor, suggesting that Plc1p is essential for glucose-induced calcium increase. This suggests that Plc1p should have a significant role in transducing glucose signal. The calcium influx induced by addition of high glucose on cells previously stimulated with low glucose levels was inhibited in strains with a deletion in the GPR1 or GPA2 genes, which suggests that glucose would be detected through the Gpr1p/Gpa2p receptor/G protein-coupled (GPCR) complex. Moreover, the signal was completely abolished in a strain unable to phosphorylate glucose, which is consistent with the reported requirement of glucose phosphorylation for GPCR complex activation.