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Nano-junctions between the endoplasmic reticulum and cytoplasmic surfaces of the plasma membrane and other organelles shape the spatiotemporal features of biological Ca2+ signals. Herein, we propose that 2D Ca2+ exchange diffusion on the negatively charged phospholipid surface lining nano-junctions participates in guiding Ca2+ from its source (channel or carrier) to its target (transport protein or enzyme). Evidence provided by in vitro Ca2+ flux experiments using an artificial phospholipid membrane is presented in support of the above proposed concept, and results from stochastic simulations of Ca2+ trajectories within nano-junctions are discussed in order to substantiate its possible requirements. Finally, we analyze recent literature on Ca2+ lipid interactions, which suggests that 2D interfacial Ca2+ diffusion may represent an important mechanism of signal transduction in biological systems characterized by high phospholipid surface to aqueous volume ratios.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root widening and aneurysm if unmanaged. We have previously reported doxycycline, a nonselective matrix metalloproteinases (MMPs) inhibitor, to attenuate aortic root widening and improve aortic contractility and elasticity in MFS mice. We were also first to use multiphoton microscopy, a non-invasive and label-free imaging technique, to quantify and link the aortic ultrastructure to possible changes in the skin dermis. Here, we aimed to assess the effects of long-term doxycycline treatment on the aortic ultrastructure and skin dermis of MFS mice through immunohistochemical evaluation and quantification of elastic and collagen content and morphology using multiphoton microscopy. Our results demonstrate a rescue of aortic elastic fiber fragmentation and disorganization accompanied by a decrease in MMP-2 and MMP-9 expression within the aortic wall in doxycycline-treated MFS mice. At 12 months of age, reduced skin dermal thickness was observed in both MFS and control mice, but only dermal thinning in MFS mice was rescued by doxycycline treatment. MMP-2 and MMP-9 expression was reduced in the skin of doxycycline-treated MFS mice. A decrease in dermal thickness was found to be positively associated with increased aortic root elastin disorganization and wall thickness. Our findings confirm the beneficial effects of doxycycline on ultrastructural properties of aortic root as well as on skin elasticity and structural integrity in MFS mice.

Aortic aneurysm is the most life-threatening complication in Marfan syndrome (MFS) patients. Doxycycline, a nonselective matrix metalloproteinases inhibitor, was reported to improve the contractile function and elastic fiber structure and organization in a Marfan mouse aorta using ex vivo small chamber myography. In this study, we assessed the hypothesis that a long-term treatment with doxycycline would reduce aortic root growth, improve aortic wall elasticity as measured by pulse wave velocity, and improve the ultrastructure of elastic fiber in the mouse model of MFS. In our study, longitudinal measurements of aortic root diameters using high-resolution ultrasound imaging display significantly decreased aortic root diameters and lower pulse wave velocity in doxycycline-treated Marfan mice starting at 6 months as compared to their non-treated MFS counterparts. In addition, at the ultrastructural level, our data show that long-term doxycycline treatment corrects the irregularities of elastic fibers within the aortic wall of Marfan mice to the levels similar to those observed in control subjects. Our findings underscore the key role of matrix metalloproteinases during the progression of aortic aneurysm, and provide new insights into the potential therapeutic value of doxycycline in blocking MFS-associated aortic aneurysm.

The present study addresses the causal relationship between induction of endo/sarcoplasmic reticulum stress and dysregulation of calcium transport, while examining whether the most widely-used experimental endo/sarcoplasmic reticulum stressors can be considered appropriate for elucidating underlying cellular mechanisms involved during the progression of the unfolded protein response in vascular smooth muscle cells. Brefeldin A is most commonly cited as inducing the stress response through an accumulation of unfolded proteins in the lumen as a result of a blockage of protein transport from the endo/sarcoplasmic reticulum to the Golgi apparatus. We investigated the effects of Brefeldin A on cellular calcium regulation during the the unfolded protein response in cultured rat vascular smooth muscle cells. Acute exposure of cells to Brefeldin A caused a small transient increase in cytoplasmic calcium, which did not cause a significant decrease in endo/sarcoplasmic reticulum calcium content. However, over the time course of 0–12 h post-treatment with Brefeldin A, we observed that the endo/sarcoplasmic reticulum of vascular smooth muscle cells exhibited an approximate fifty percent decrease in calcium concentration after the first hour of exposure, which is maintained over the next eleven hours, whereas concentrations of unfolded protein response markers only began to increase significantly around nine to twelve hours post-treatment. We have concluded that the endo/sarcoplasmic reticulum calcium drop, which up to now, has been considered as a characteristic of the late onset of cellular stress response, occurs prior to the initiation of the unfolded protein response, rather than as a result of its many corrective pathways.

The fertility of men with neurofibromatosis 1 (NF1) is reduced. Despite this observation, gonadal function has not been examined in patients with NF1. In order to assess the role of reduced neurofibromin in the testes, we examined testicular morphology and function in an Nf1+/- mouse model. We found that although Nf1+/- male mice are able to reproduce, they have significantly fewer pups per litter than Nf1+/+ control males. Reduced fertility in Nf1+/- male mice is associated with disorganization of the seminiferous epithelium, with exfoliation of germ cells and immature spermatids into the tubule lumen. Morphometric analysis shows that these alterations are associated with decreased Leydig cell numbers and increased spermatid cell numbers. We hypothesized that hyper-activation of Ras in Nf1+/- males affects ectoplasmic specialization, a Sertoli-spermatid adherens junction involved in spermiation. Consistent with this idea, we found increased expression of phosphorylated ERK, a downstream effector of Ras that has been shown to alter ectoplasmic specialization, in Nf1+/- males in comparison to control Nf1+/+ littermates. These data demonstrate that neurofibromin haploinsufficiency impairs spermatogenesis and fertility in a mouse model of NF1.

In this chapter we examine the importance of cytoplasmic nanojunctions-nanometer scale appositions between organellar membranes including the molecular transporters therein-to the cell signaling machinery, with specific reference to Ca

Regular low-impact physical activity is generally allowed in patients with Marfan syndrome, a connective tissue disorder caused by heterozygous mutations in the fibrillin-1 gene. However, being above average in height encourages young adults with this syndrome to engage in high-intensity contact sports, which unfortunately increases the risk for aortic aneurysm and rupture, the leading cause of death in Marfan syndrome. In this study, we investigated the effects of voluntary (cage-wheel) or forced (treadmill) aerobic exercise at different intensities on aortic function and structure in a mouse model of Marfan syndrome. Four-week-old Marfan and wild-type mice were subjected to voluntary and forced exercise regimens or sedentary lifestyle for 5 mo. Thoracic aortic tissue was isolated and subjected to structural and functional studies. Our data showed that exercise improved aortic wall structure and function in Marfan mice and that the beneficial effect was biphasic, with an optimum at low intensity exercise (55–65% V̇o2max) and tapering off at a higher intensity of exercise (85% V̇o2max). The mechanism underlying the reduced elastin fragmentation in Marfan mice involved reduction of the expression of matrix metalloproteinases 2 and 9 within the aortic wall. These findings present the first evidence of potential beneficial effects of mild exercise on the structural integrity of the aortic wall in Marfan syndrome associated aneurysm. Our finding that moderate, but not strenuous, exercise protects aortic structure and function in a mouse model of Marfan syndrome could have important implications for the medical care of young Marfan patients. NEW & NOTEWORTHY The present study provides conclusive scientific evidence that daily exercise can improve aortic health in a mouse model of Marfan syndrome associated aortic aneurysm, and it establishes the threshold for the exercise intensity beyond which exercise may not be as protective. These findings establish a platform for a new focus on promoting regular exercise in Marfan patients at an optimum intensity and create a paradigm shift in clinical care of Marfan patients suffering from aortic aneurysm complications.

Maintenance of steady-state calcium (Ca(2+)) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca(2+) content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca(2+) transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca(2+)-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca(2+) indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca(2+) store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca(2+). This tool is useful for investigation into the nuanced changes in SR Ca(2+) that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca(2+) content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca(2+) indicator, D1SR, is used to detect Ca(2+) fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca(2+) levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca(2+) movement.

The couplons of the cardiomyocyte form nanospaces within the cell that place the L-type calcium channel (Ca(v)1.2), situated on the plasmalemma, in opposition to the type 2 ryanodine receptor (RyR2), situated on the sarcoplasmic reticulum. These two molecules, which form the basis of excitation-contraction coupling, are separated by a very limited space, which allows a few Ca(2+) ions passing through Ca(v)1.2 to activate the RyR2 at concentration levels that would be deleterious to the whole cell. The limited space also allows Ca(2+) inactivation of Ca(v)1.2. We have found that not all couplons are the same and that their properties are likely determined by their molecular partners which, in turn, determine their excitability. In particular, there are a class of couplons that lie outside the RyR2-Ca(v)1.2 dyad; in this case, the RyR2 is close to caveolin-3 rather than Ca(v)1.2. These extra-dyadic couplons are probably controlled by the multitude of molecules associated with caveolin-3 and may modulate contractile force under situations such as stress. It has long been assumed that like the skeletal muscle, the RyR2 in the couplon are arranged in a structured array with the RyR2 interacting with each other via domain 6 of the RyR2 molecule. This arrangement was thought to provide local control of RyR2 excitability. Using 3D electron tomography of the couplon, we show that the RyR2 in the couplon do not form an ordered pattern, but are scattered throughout it. Relatively few are in a checkerboard pattern--many RyR2 sit edge-to-edge, a configuration which might preclude their controlling each other's excitability. The discovery of this structure makes many models of cardiac couplon function moot and is a current avenue of further research.

Traffic related particulate matter air pollution is a risk factor for cardiovascular events; however, the biological mechanisms are unclear. We hypothesize that diesel exhaust (DE) inhalation induces up-regulation of inducible nitric oxide synthase (iNOS), which is known to contribute to vascular dysfunction, progression of atherosclerosis and ultimately cardiovascular morbidity and mortality. Methods: ApoE knockout mice (30-week) were exposed to DE (at 200 {mu}g/m{sup 3} of particulate matter) or filtered-air (control) for 7 weeks (6 h/day, 5 days/week). iNOS expression in the blood vessels and heart was evaluated by immunohistochemistry and western blotting analysis. To examine iNOS activity, thoracic aortae were mounted in a wire myograph, and vasoconstriction stimulated by phenylephrine (PE) was measured with and without the presence of the specific inhibitor for iNOS (1400 W). NF-{kappa}B (p65) activity was examined by ELISA. The mRNA expression of iNOS and NF-{kappa}B (p65) was determined by real-time PCR. Results: DE exposure significantly enhanced iNOS expression in the thoracic aorta (4-fold) and heart (1.5 fold). DE exposure significantly attenuated PE-stimulated vasoconstriction by {approx} 20%, which was partly reversed by 1400 W. The mRNA expression of iNOS and NF-{kappa}B was significantly augmented after DE exposure. NF-{kappa}B activity was enhanced 2-fold after DE inhalation, andmore » the augmented NF-{kappa}B activity was positively correlated with iNOS expression (R{sup 2} = 0.5998). Conclusions: We show that exposure to DE increases iNOS expression and activity possibly via NF-{kappa}B-mediated pathway. We suspect that DE exposure-caused up-regulation of iNOS contributes to vascular dysfunction and atherogenesis, which could ultimately lead to urban air pollution-associated cardiovascular morbidity and mortality. - Highlights: > Exposed ApoE knockout mice (30-week) to diesel exhaust (DE) for 7 weeks. > Examine iNOS expression and activity in the blood vessels and heart. > DE exposure enhanced iNOS protein and mRNA expression in the aorta and heart. > iNOS activity was also increased after DE exposure. > This up-regulation of iNOS may contribute to vascular dysfunction and atherogenesis.« less

In this study, we compared the potencies of diosgenin, a plant-derived sapogenin structurally similar to estrogen and progesterone, on vascular smooth muscle functions ranging from contraction and migration to apoptosis. The effects of diosgenin on vascular smooth muscle cell viability and migration were measured using a primary mouse aortic smooth muscle cell culture. The effects of diosgenin on smooth muscle cell contraction and calcium signaling were investigated in the isolated mouse aorta using wire myography and confocal microscopy, respectively. Here, we report that in cultured cells diosgenin (≥ 25 μM) induces apoptosis as measured by the number of annexin V-positive cells and caspase-3 cleavage, while decreasing cell viability as indicated by protein kinase B/Akt phosphorylation. In addition, diosgenin blocks smooth muscle cell migration in a transwell Boyden chamber in response to serum treatment and response to injury in a cell culture system. Diosgenin (≥ 25 μM) also significantly blocks receptor-mediated calcium signals and smooth muscle contraction in the isolated aorta. There is no difference in the inhibitory effects of diosgenin on vascular smooth muscle contraction between the endothelium-intact and endothelium-denuded aortic segments, indicating that they are caused by altered smooth muscle activity. Our findings suggest that over the concentration range of 10 to 15 μM diosgenin may provide overall beneficial effects on diseased vascular smooth muscle cells by blocking migration and contraction without any significant cytopathic effects, implying a potential therapeutic value for diosgenin in vascular disorders.

Subplasmalemmal ion fluxes have global effects on Ca 2+ signaling in vascular smooth muscle. Measuring cytoplasmic and mitochondrial [Ca 2+ ]and [Na + ], we previously showed that mitochondria buffer both subplasmalemmal cytosolic [Ca 2+ ] and [Na + ] in vascular smooth muscle cells. We have now directly measured sarcoplasmic reticulum [Ca 2+ ] in aortic smooth muscle cells, revealing that mitochondrial Na + /Ca 2+ exchanger inhibition with CGP-37157 impairs sarcoplasmic reticulum Ca 2+ refilling during purinergic stimulation. By overexpressing hFis1 to remove mitochondria from the subplasmalemmal space, we show that the rate and extent of sarcoplasmic reticulum refilling is augmented by a subpopulation of peripheral mitochondria. In ATP-stimulated cells, hFis-1–mediated relocalization of mitochondria impaired the sarcoplasmic reticulum refilling process and reduced mitochondrial [Ca 2+ ] elevations, despite increased cytosolic [Ca 2+ ] elevations. Reversal of plasmalemmal Na + /Ca 2+ exchange was the primary Ca 2+ entry mechanism following ATP stimulation, based on the effects of KB-R7943. We propose that subplasmalemmal mitochondria ensure efficient sarcoplasmic reticulum refilling by cooperating with the plasmalemmal Na + /Ca 2+ exchanger to funnel Ca 2+ into the sarcoplasmic reticulum and minimize cytosolic [Ca 2+ ] elevations that might otherwise contribute to hypertensive or proliferative vasculopathies.

Background: Occurrence of disease complications in the abdominal aorta in Marfan syndrome, a connective tissue disorder caused by mutations in the gene encoding fibrillin-1, is relatively rare. We hypothesized that Marfan syndrome could affect the structure, vasomotor function and mechanical property of the abdominal aorta. Methods and Results: Abdominal aorta from mice at 3, 6, 9 and 12 months of age, heterozygous for the Fbn1 allele encoding a cysteine substitution (Fbn1C1039G/+, Marfan mice, n = 50), were compared with those from age-matched control littermates (n = 50). Marfan abdominal aorta demonstrated pronounced elastic fiber degradation and disorganization, concomitant with an increased aortic stiffness during aging. In the isometric force measurement, vasoconstriction in response to membrane depolarization or phenylephrine stimulation was similar in both Marfan and control abdominal aorta. However, Marfan abdominal aorta was less sensitive to the inhibition of the phenylephrine-induced contraction by indomethacin and SQ-29548, during which the release of thromboxane A2 was one half of that of the controls. Nevertheless, the protein expression of cyclooxygenase-1 and cyclooxygenase-2 detected by Western immunoblotting was not different between the 2 strains. Conclusions: We demonstrated that Marfan syndrome affected abdominal aorta with respect to matrix elastic fiber organization, aortic stiffness and release of thromboxane A2.

Background Vein arterialization following bypass surgery often leads to graft occlusion, but the underlying cellular mechanisms have been poorly studied. Objectives Cell cycle progression and the activation of proliferation signalling were compared in arterialized grafts prepared either according to the conventional procedure or using pharmacological relaxation with the native vein. Methods Using the porcine carotid-jugular bilateral interposition graft model on one side, a segment of porcine jugular vein was prepared for grafting using the conventional procedure, with pressure distention at 300 mmHg; the segment grafted on the other side was treated with a combination of pharmacological vasodilators. Both veins were grafted into the carotid artery for two weeks. Results On the immunolabelling of proliferation cell nuclear antigen, a greater number of proliferating cells was found in the conventionally prepared grafts compared with pharmacologically prepared grafts. Cyclin D1 expression and phosphorylation of retinoblastoma increased after implantation, coinciding with nuclear accumulation of beta-catenin, activation of the Akt and mitogen activated protein kinase cascades, and upregulated phosphatase and tensin homologue phosphorylation. Replacement of distention with pharmacological relaxation reduced the increase in cyclin D1 expression, phosphorylation of retinoblastoma, Akt-Thr 308 , glycogen synthase kinase 3 beta and p38, but not extracellular signal-regulated kinases. This technique preserved the active phosphatase and tensin homologue, as well as the expression of cyclin-dependent kinase inhibitor p21 Cip1 , while elevating the expression of p27 Kip1 . Conclusions It was concluded that two-week arterial implantation stimulates proliferation signalling and promotes the cell cycle in vein grafts. Replacement of the conventional preparation procedures with pharmacological vasorelaxation restricts the activation of proliferation and cell cycle progression, and can be beneficial for improving vein graft patency.

Reversal of the Na + /Ca 2+ -exchanger (NCX) has been shown to mediate Ca 2+ influx during activation of G-protein linked receptors. Functional coupling between the reverse-mode NCX and the canonical transient receptor potential channels (TRPCs) has been proposed to mediate Ca 2+ influx in HEK-293 cells overexpressing TRPC3. In this communication we present evidence for similar functional coupling of NCX to endogenously expressed TRPC6 in rat aorta smooth muscle cells. Selective inhibition of reverse-mode NCX with KB-R7943 and of non-selective cation-channels with SKF-96365 abolished Ca 2+ influx in response to agonist stimulation (ATP). Expression of a dominant negative TRPC6 mutant also reduced the Ca 2+ influx in proportion to its transfection efficiency. Calyculin A, which is known to disrupt the junctions of the plasma membrane and sarco/endoplasmic reticulum, increased global Na + elevations and reduced stimulated Ca 2+ influx. Together our data provide evidence that localized Na + elevations are generated by TRPC6 and drive reversal of NCX to mediate Ca 2+ influx.

Here we show that male, but not female mice lacking expression of the GTPase M-Ras developed urinary retention with distention of the bladder that exacerbated with age but occurred in the absence of obvious anatomical outlet obstruction. There were changes in detrusor morphology in Mras -/- males: Smooth muscle tissue, which exhibited a compact organization in WT mice, appeared disorganized and became increasingly ‘layered’ with age in Mras -/- males, but was not fibrotic. Bladder tissue near the apex of bladders of Mras -/- males exhibited hypercontractility in response to the cholinergic agonist carbachol in in vitro, while responses in Mras -/- females were normal. In addition, spontaneous phasic contractions of detrusors from Mras -/- males were increased, and Mras -/- males exhibited urinary incontinence. We found that expression of the muscarinic M2 and M3 receptors that mediate the cholinergic contractile stimuli of the detrusor muscle was dysregulated in both Mras -/- males and females, although only males exhibited a urinary phenotype. Elevated expression of M2R in young males lacking M-Ras and failure to upregulate M3R with age resulted in significantly lower ratios of M3R/M2R expression that correlated with the bladder abnormalities. Our data suggests that M-Ras and M3R are functionally linked and that M-Ras is an important regulator of male bladder control in mice. Our observations also support the notion that bladder control is sexually dimorphic and is regulated through mechanisms that are largely independent of acetylcholine signaling in female mice.

Stimulation of the tracheal muscle bundle by acetylcholine (ACh) results in the generation of asynchronous repetitive Ca2+ waves (ACW) in intact tracheal smooth muscle (TSM) cells. We showed previously that ACW underlie cholinergic excitation-contraction coupling in porcine TSM and that Ca2+ entry through the L-type voltage-gated Ca2+ channel (VGCC) contributes partially to maintenance of the ACW. However, the mechanism of the ACW remains undefined. In this study, we pharmacologically characterized the mechanism of ACh-induced ACW in the intact porcine tracheal muscle bundle. We found that inhibition of receptor-operated channels/store-operated channels (ROC/SOC) by SKF-96365 completely abolished the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Blockade of Na+/Ca2+ exchange with KB-R7943 or 2′,4′-dichlorobenzamil or removal of extracellular Na+ resulted in nearly complete inhibition of the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase by cyclopiazonic acid abolished the ongoing ACW. Application of 2-aminoethoxydiphenyl borate (2-APB) or xestospongin C to inhibit the inositol 1,4,5-trisphosphate-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels produced no effect on ACh-mediated ACW and tonic contraction. However, pretreatment with caffeine or ryanodine inhibited ACh-induced ACW. Furthermore, application of procaine or tetracaine prevented the generation and abolished the ongoing ACh-mediated ACW and tonic contraction. Collectively, these results indicate that the ACh-stimulated ACW in porcine TSM are produced by repetitive cycles of Ca2+ release from SR through 2-APB- and xestospongin C-insensitive Ca2+ release channels, and plasmalemmal Ca2+ entry involving reverse-mode Na+/Ca2+ exchange, ROC/SOC, and L-type VGCC is required to refill the SR via SERCA to support the ongoing ACW.

ObjectiveAutogenous vein bypasses are a common and effective method to treat occlusive disease. During surgical preparation, veins are routinely pressure distended to overcome vasospasm and twists. Distention, however, is believed to promote vascular remodeling and contribute to decreased graft patency. Pharmacologic vasorelaxation with a combination of effective vasodilators has been suggested as an alternative to pressure distention. The extracellular matrix (ECM)-degrading matrix metalloproteinases (MMPs) have been implicated in vascular remodeling and neointima formation. The purpose of the present study was to compare the effects of pressure distention with pharmacologic vasorelaxation on graft remodeling and regulation of MMP-2 and MMP-9 in porcine vein grafts.MethodsCarotid artery bypass utilizing internal jugular veins was performed in eight female white pigs. Jugular veins were randomized to receive pressure distention (300 mm Hg for 2 minutes) or a combination of vasodilators (the α-adrenergic antagonist phenoxybenzamine, 10 μmol/L; the Rho-kinase inhibitor HA-1077 [fasudil], 50 μmol/L; and the calcium-channel blocker nicardipine, 1 μmol/L) for 30 minutes and then were grafted into the carotid arteries. Two weeks after surgery, vein graft samples were analyzed for vessel intimal and medial area, lumen diameter, and ECM composition. Molecular analysis using reverse transcription-polymerase chain reaction, Western immunoblotting, gelatin zymography, and reverse zymography were performed to study the expression and activation of MMP-2 and MMP-9, and tissue inhibitors of MMP (TIMP)-1 and TIMP-2.ResultsPressure distention irreversibly overstretched the porcine jugular vein and increased MMP-2 and MMP-9 proteolytic activity by 40% and 77%, respectively. Two weeks of vein grafting in the carotid arterial bed induced vessel wall thickening, ECM modification, and neointima formation, which were more pronounced in the distended grafts (P < .05) and accompanied by an increase in MMP expression and activity. Distended grafts demonstrated higher percentages of active MMP-9 (17.8% ± 1.0%) and higher activities of latent (35.5% ± 3.3%) and active MMP-2 (69.6% ± 8.8%) than the pharmacologically treated grafts. Protein expression of TIMP-1 and TIMP-2 was downregulated after arterial grafting, but the pharmacologically treated grafts expressed significantly more TIMP-1 protein (by 36.8% ± 4.1%) than the distended ones. The activities of TIMPs were markedly decreased after grafting, contributing to the upregulated MMP activity.ConclusionsPressure distention of vein grafts before implantation, compared with pharmacologic vasodilatation, stimulates neointima formation and augments MMP activities. Pharmacologic vasorelaxation may be clinically superior to distention in attenuating graft remodeling and possibly improving graft patency.Clinical RelevanceAutogenous vein bypasses are a common and effective method to treat occlusive disease. This study demonstrated that pressure distention, a common preparatory procedure in bypass surgery, upregulates extracellular matrix-degrading matrix metalloproteinases, which predisposes vein grafts to extensive remodeling and contributes to neointima formation and graft occlusion. The topical application of a combination of vasodilators to the vein graft before implantation may be clinically superior to pressure distention in attenuating graft remodeling and may possibly improve graft patency and reduce secondary surgical interventions.

Asynchronous Ca2+ waves or wave-like [Ca2+]i oscillations constitute a specialized form of agonist-induced Ca2+ signaling that is observed in a variety of smooth muscle cell types. Functionally, it is involved in the contractile regulation of the smooth muscle cells as it signals for tonic contraction in certain smooth muscle cells while causing relaxation in others. Mechanistically, repetitive Ca2+ waves are produced by repetitive cycles of sarcoplasmic reticulum Ca2+ release followed by Ca2+ uptake. Plasmalemmal Ca2+ entry mechanisms are important for providing the additional Ca2+ necessary to maintain proper refilling of the sarcoplasmic reticulum Ca2+ store and support ongoing Ca2+ waves. In this paper, we will review the phenomenon of asynchronous Ca2+ waves in smooth muscle and discuss the scientific and clinical significance of this new understanding.Key words: excitation-contraction coupling, confocal fluoresence microscopy, calcium signaling.

The mitochondria and the sarcoplasmic reticulum (SR) are two major intracellular calcium-storing organelles that exhibit close functional interaction with each other. Close spatial association is believed to be important for their functional interaction. In this study, we have characterized the spatial relationship between the SR and the mitochondria in porcine tracheal smooth muscle cells (TSMC) under different conditions. By examining the cross-section of unstimulated TSMC with electron microscopy, we found that 99.4 +/- 0.5% of the mitochondria seen on random cross-sections were situated within 30 nm of the SR and that 82.2 +/- 6.7% of the mitochondria were completely enveloped by the SR network. Overall, 48.0 +/- 3.5% of the mitochondrial outer membrane was within 30 nm with the SR. After stimulation of the TSMC with acetylcholine (ACh) or 80 mM [K(+)] solution 97.0 +/- 2.1% and 98.6 +/- 1.4% of the mitochondria observed were situated within 30 nm of the SR, respectively. However, the proportion of the mitochondria that was completely enveloped by the SR was significantly reduced to 12.2 +/- 5.9% in ACh-stimulated cells and 9.7 +/- 6.6% in 80 mM [K(+)] stimulated cells. The percentage of mitochondrial membrane closely associated with the SR was correspondingly lower at 10.1 +/- 1.0% during ACh stimulation and 10.8 +/- 0.9% during 80 mM [K(+)] stimulation. During smooth muscle cell stimulation, the SR appears to unwrap from the mitochondria and extend into the cytoplasm while maintaining close contact with the mitochondria over a smaller area. Such static and dynamic components of the close spatial association between the mitochondria and the SR may serve as a structural basis for the selective and efficient Ca(2+) trafficking between the two organelles in TSMC.

Excitation–contraction coupling (E–C coupling) in phenylephrine(PE)-stimulated rabbit inferior vena cava (IVC) depends on the generation of asynchronous recurring Ca 2+ waves in the in situ vascular smooth muscle cells (VSMC). Previous studies by our group have implicated a putative non-selective cationic store-operated channel and the reverse-mode Na + –Ca 2+ exchange in refilling of the intracellular Ca 2+ store via the sarco/endoplasmic reticulum ATPase (SERCA) and the maintenance of the recurring Ca 2+ waves. We hypothesize that for the proper functioning of these three Ca 2+ translocators in the process of SR refilling, the plasma membrane (PM) and the underlying superficial sarcoplasmic reticulum (SR) form specialized PM–SR junctions, which are essential for the maintenance of the recurring Ca 2+ waves. In order to test this hypothesis, calyculin-A, a serine/threonine phosphatase inhibitor that has been demonstrated to result in the disruption of the PM–SR junctions was used. In the control rabbit IVC, electron microscopy of the in situ VSMC indicates that 14.2 ± 0.7% of the PM is closely apposed by the prominent superficial SR network, forming numerous flattened PM–SR junctional cytoplasmic spaces. In the control IVC stimulation with 5 μM PE resulted in sustained recurring Ca 2+ waves with a frequency of 0.42 ± 0.02 Hz. In calyculin-A treated rabbit IVC, a concentration-dependent dissociation of the superficial SR and loss of PM–SR junctions was observed. This progressive loss of the PM–SR junctions occurs over the same concentration range as the inhibition of PE-induced recurring Ca 2+ waves. These findings offer support for the hypothesis that the presence of the PM–SR junctions is required for the generation of asynchronous recurring Ca 2+ waves, which underlie excitation–contraction coupling in the VSMC of the rabbit IVC.

Fluctuations in intracellular calcium concentration ([Ca2+]i) constitute the main link in excitation-contraction coupling (E-C coupling) in airway smooth muscle cells (ASMC). It has recently been reported that ACh induces asynchronous recurring Ca2+ waves in intact ASMC of murine bronchioles. With the use of a novel technique allowing us to simultaneously measure subcellular [Ca2+]i and force generation in ASMC located within an intact tracheal muscle bundle, we examined a similar pattern of Ca2+ signaling in the trachea. We found that application of ACh resulted in the generation of recurring intracellular Ca2+ waves progressing along the longitudinal axis of the ribbon-shaped intact ASMC. These Ca2+ waves were not synchronized between neighboring cells, and induction of wave-like [Ca2+]i oscillations was temporally associated with development of force by the tracheal muscle bundle. By comparing the concentration dependence of force generation and the parameters characterizing the [Ca2+]i oscillations, we found that the concentration-dependent increase in ACh-induced force development by the tracheal smooth muscle bundle is achieved by differential recruitment of intact ASMC to initiate Ca2+ waves and by enhancement in the frequency of [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. Our findings demonstrate that asynchronous recurring Ca2+ waves underlie E-C coupling in ACh-induced contraction of the intact tracheal smooth muscle bundle. Furthermore, in contrast to what was reported in enzymatically dissociated ASMC, Ca2+ influx through the L-type voltage-gated Ca2+ channel was not an obligatory requirement for the generation of [Ca2+]i oscillations and development of force in ACh-stimulated intact ASMC.

We developed an in situ assay system to simultaneously monitor intracellular Ca2+concentration ([Ca2+]i, fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 μM) stimulated a rapid increase in [Ca2+]ifollowed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 μM) induced a gradual, small increase in [Ca2+]iand a slow increase in intracellular NO levels. Removal of extracellular Ca2+and depletion of Ca2+stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca2+]iby application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca2+]iand intracellular NO production in the intact endothelium is a powerful tool to study Ca2+-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca2+in tyrosine phosphorylation-induced NO production.

Vascular smooth muscle shows both plasticity and heterogeneity with respect to Ca2+signaling. Physiological perturbations in cytoplasmic Ca2+concentration ([Ca2+]i) may take the form of a uniform maintained rise, a transient uniform [Ca2+]ielevation, a transient localized rise in [Ca2+]i(also known as spark and puff), a transient propagated wave of localized [Ca2+]ielevation (Ca2+wave), recurring asynchronous Ca2+waves, or recurring synchronized Ca2+waves dependent on the type of blood vessel and the nature of stimulation. In this overview, evidence is presented which demonstrates that interactions of ion transporters located in the membranes of the cell, sarcoplasmic reticulum, and mitochondria form the basis of this plasticity of Ca2+signaling. We focus in particular on how the junctional complexes of plasmalemma and superficial sarcoplasmic reticulum, through the generation of local cytoplasmic Ca2+gradients, maintain [Ca2+]ioscillations, couple these to either contraction or relaxation, and promote Ca2+cycling during homeostasis.

α1-Aderenoceptor-mediated constriction of rabbit inferior vena cava (IVC) is signaled by asynchronous wavelike Ca2+ oscillations in the in situ smooth muscle. We have shown previously that a putative nonselective cationic channel (NSCC) is required for these oscillations. In this report, we show that the application of 2-aminoethoxyphenyl borate (2-APB) to antagonize inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ release channels (IP3R channels) can prevent the initiation and abolish ongoing α1-aderenoceptor-mediated tonic constriction of the venous smooth muscle by inhibiting the generation of these intracellular Ca2+ concentration ([Ca2+]i) oscillations. The observed effects of 2-APB can only be attributed to its selective inhibition on the IP3R channels, not to its slight inhibition of the L-type voltage-gated Ca2+ channel and the sarco(endo)plasmic reticulum Ca2+ ATPase. Furthermore, 2-APB had no effect on the ryanodine-sensitive Ca2+ release channel and the store-operated channel (SOC) in the IVC. These results indicate that the putative NSCC involved in refilling the sarcoplasmic reticulum (SR) and maintaining the tonic contraction is most likely an SOC-type channel because it appears to be activated by IP3R-channel-mediated SR Ca2+ release or store depletion. This is in accordance with its sensitivity to Ni2+ and La3+ (SOC blockers). More interestingly, RT-PCR analysis indicates that transient receptor potential (Trp1) mRNA is strongly expressed in the rabbit IVC. The Trp1 gene is known to encode a component of the store-operated NSCC. These new data suggest that the activation of both the IP3R channels and the SOC are required for PE-mediated [Ca2+]i oscillations and constriction of the rabbit IVC.

We report the modulatory effects of chronic oral LY139481 (raloxifene) on basal release of nitric oxide (NO) and mRNA levels of endothelial NO synthase (eNOS) in rat thoracic aorta. Constrictor dose-response curves to phenylephrine were generated before and after pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Aortic segments were obtained from four groups of rats gavaged orally for 21 days: (i) ovariectomized, (ii) sham, (iii) ovariectomized estradiol-treated, and (iv) ovariectomized raloxifene-treated. Intact aortic rings from sham rats and ovariectomized rats receiving raloxifene and estrogen showed a greater potentiation of the phenylephrine responses after L-NAME. Semi-quantitative reverse transcription-polymerase chain reaction indicated a gender-based difference in eNOS mRNA expression in thoracic aorta. Moreover, we demonstrated that eNOS mRNA expression in the upper thoracic aorta was significantly higher in treatment groups. These results show that chronically administered raloxifene is exerting a potentially important vasculo-protective effect by stimulating eNOS expression.

In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.

We investigated the role of protein kinase C (PKC) isoforms on changes in sensitivity of contractile mechanisms to intracellular Ca(2+) (force /[Ca(2+)]i) by phenylephrine (0.1-100 microM) in rat tail arterial helical strips using simultaneous measurements of force and [Ca(2+)]i. Force/[Ca(2+)]Ii induced by phenylephrine was greater than that induced by 80 mM K+. Force/[Ca(2+)]i induced by phenylephrine in physiologic saline solution or low Ca(2+) solution was dependent on the agonist concentration. Removal of Ca(2+) completely abolished the phenylephrine-induced contraction. The PKC inhibitors staurosporine and calphostin C inhibited the increase in force/[Ca(2+)]i induced by phenylephrine to a much greater extent than that induced by 80 mM K+. LY379196, a specific PKCbeta inhibitor, did not inhibit the increase of calcium sensitivity due to phenylephrine. The classic PKC isoforms, alpha, betaI, and II not gamma were demonstrated in the artery by immunohistochemistry. These results suggest that in rat tail arterial smooth muscle, PKCalpha, and not beta or gamma, mediates the increase of changes in sensitivity of contractile mechanisms to intracellular Ca(2+) to high dose of alpha1 receptor stimulation (phenylephrine 100 microM) on nonphysiologic conditions.

Background —The physiological consequences of inducible NO synthase (iNOS) expression were studied in allograft coronary arteries by pressure myography. Methods and Results —Septal coronary arteries (diameter, 200.6±3.3 μm) were harvested from allograft and isograft hearts, and their myogenic properties were measured before and after iNOS and nonselective NOS inhibition with aminoguanidine (AG, 100 μmol/L) and N G -nitro- l -arginine methyl ester (L-NAME) (200 μmol/L). Fura 2 fluorescence microscopy was used to measure [Ca 2+ ] i in isolated endothelial cells. Monoclonal anti-iNOS immunostains demonstrated iNOS protein in day 2, 7, 14, and 28 allograft vessels, but only in day 2 isograft vessels. Myogenic tone was profoundly inhibited in allograft vessels from day 4 onward. In day 4 allograft vessels, these differences were abolished by L-NAME but not AG, suggesting greater basal release of eNOS-based NO from allograft endothelium. Fluorescence measurements confirmed elevation of [Ca 2+ ] i in day 4 allograft endothelium, providing a mechanism for enhanced eNOS activity. For days 7 to 28, AG potentiated myogenic tone in allograft but not isograft vessels, indicating that vasoactive iNOS-based NO was present. In mature vessels, constriction via agonist- and depolarization-mediated mechanisms showed parallel inhibition, suggesting an intrinsic defect in vascular smooth muscle cell contraction. Conclusions —Our data indicate that the profound inhibition of myogenic tone in allograft arteries involves direct vasodilation by eNOS- and iNOS-based NO, as well as an intrinsic defect in vascular smooth muscle contraction. The hemodynamic profile resulting from these changes in allograft resistance vessel function would favor movement of extracellular fluid from the intravascular space into the myocardial interstitium, resulting in edema, increased ventricular stiffness, and poor ventricular performance.

Activation of MAP kinase kinase, also called ERK kinase (MEK), may lead to desinhibition of thin filament regulatory proteins and we therefore investigated the acute effects of the potent MEK inhibitor, PD98059 on the contractile properties of pressurized rat middle cerebral arteries. Cerebral arteries (diameter 100-150 microm) were mounted on a pressure myograph and PD98059 (10 microM, 40 microM) significantly inhibited (15% and 64%) myogenic tone (P < 0.001). At these concentrations, PD98059 also significantly reduced the vasopressin (0.1 microM)- and KCl (60 mM)-induced tone. Cumulative addition of exogenous Ca2+ (0.4-1.6 mM) increased myogenic tone to approximately 50% of constriction at 80 mmHg. This effect was inhibited by PD98059 (P < 0.001). These results demonstrate that pressure-induced myogenic tone is inhibited by PD98059 at the concentrations that have been reported to be selective for inhibition of MEK and the MAP kinase cascade. However, our results also demonstrate that PD98059 may have nonspecific effects on voltage-sensitive Ca2+ entry in vascular smooth muscle.

Ca2+extrusion from rabbit inferior vena cava smooth muscle was studied using ratiometric fura 2 fluorimetry. Concomitant blockade of the plasma membrane Ca2+-adenosinetriphosphatase (ATPase; PCMA), Na+-Ca2+exchanger, and sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) completely prevented the decline in intracellular Ca2+concentration ([Ca2+]i) normally observed when Ca2+is removed from the extracellular space (ECS) after stimulated Ca2+influx. Blockade of the Na+-Ca2+exchanger by removal of external Na+reduced the rate of [Ca2+]idecline by 47%. Blockade of SERCA with cyclopiazonic acid reduced it by 23%, and this was not additive to the effects of Na+removal. Exposure to nominally Ca2+-free solution prevented the sarcoplasmic reticulum (SR) from reloading only if the Na+-Ca2+exchanger was operational. Our results can be explained by an SR contribution to Ca2+extrusion in which SERCA is arranged in series with Na+-Ca2+exchange.

The purpose of the present investigation was to examine the correlation between rates of endoplasmic reticulum (ER) depletion and activation of store operated channels (SOC) in freshly isolated rabbit aortic endothelial cells. We investigated the effects of 10μM cyclopiazonic acid (CPA), 10μM ryanodine, and 10mM caffeine on the rate of Ca 2+ depletion from the ER and on Ca 2+ and Mn 2+ influx using fura-2 fluorescence. We observed that the spontaneous loss of the ACh-sensitive pool is slow. Activation of ryanodine receptors (caffeine, ryanodine) or inhibition of the ER Ca 2+ pump (CPA) increased the rate of Ca 2+ loss from the ACh-sensitive pool. CPA stimulated Mn 2+ influx, while caffeine and ryanodine did not. Our results show non linear correlation between ER depletion and activation of divalent cation entry. In the case of CPA, less than 20% depletion of the ACh sensitive store was required for full activation of SOC, while caffeine and ryanodine deplete over 50% of the ACh sensitive store without activating any influx. These data suggest that only a small compartment of the ER is involved in regulation of Ca 2+ entry.

Agonist-stimulated smooth muscle Ca2+ waves regulate blood vessel tone and vasomotion. Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were stimulated by a combination of inositol 1,4,5-trisphosphate- and Ca2+ -induced Ca2+ release from the endo/sarcoplasmic reticulum. Herein, we present the first report of endothelin-1 stimulated waves of Ca2+ depletion from the sarcoplasmic reticulum of vascular smooth muscle cells using a calsequestrin-targeted Ca2+ indicator. Our findings confirm that these waves are due to regenerative Ca2+ -induced Ca2+ release by the receptors for inositol 1,4,5-trisphosphate. Our main new finding is a transient elevation in SR luminal Ca2+ concentration ([Ca2+](SR)) both at the site of wave initiation, just before regenerative Ca2+ release commences, and at the advancing wave front, during propagation. This strongly suggests a role for [Ca2+](SR) in the activation of inositol 1,4,5-trisphosphate receptors during agonist-induced calcium waves. In addition, quantitative analysis of the gradual decrease in the velocity of the depletion wave, observed in the absence of external Ca2+, indicates continuity of the lumen of the sarcoplasmic reticulum network. Finally, our observation that the depletion wave was arrested by the nuclear envelope may have implications for selective Ca2+ signalling.

Abstract In a freshly isolated endothelial cell preparation from rabbit aorta, the regulation of the acetylcholine (ACh)–sensitive intracellular Ca 2+ store and the effects of the Ca 2+ -induced Ca 2+ release agonists ryanodine and caffeine were studied using fura 2 imaging fluorescence microscopy. ACh (10 μmol/L) caused a transient release of Ca 2+ from an intracellular store, presumably via an inositol tris-phosphate–sensitive mechanism. This ACh response could be repeated in the presence of extracellular Ca 2+ but was obtained only once in Ca 2+ -free bathing solution, which shows that a depleted intracellular Ca 2+ store can be rapidly refilled from the extracellular space. Refilling can be prevented by the endoplasmic reticulum Ca 2+ -ATPase inhibitor cyclopiazonic acid (10 μmol/L), implying that Ca 2+ enters the cytoplasm before accumulation in the endoplasmic reticulum. Ionomycin (10 μmol/L) caused a large Ca 2+ release even after the ACh-releasable store had been emptied, indicating the existence of other ACh-insensitive stores, perhaps including the mitochondria. In one third of the cells studied, ACh induced oscillations in [Ca 2+ ] i that were dependent on extracellular Ca 2+ . Also investigated were the effects of caffeine and ryanodine. In this cell preparation neither caffeine nor ryanodine induced a Ca 2+ transient but instead slowly increased [Ca 2+ ] i . It was observed that both caffeine and ryanodine were able to slowly deplete the ACh-sensitive store. These results indicate the presence of functional ryanodine receptors in native endothelial cells and demonstrate overlap between the caffeine and agonist-sensitive Ca 2+ stores. We also found that caffeine was able to directly inhibit the process of ACh-induced Ca 2+ release. It is hypothesized that endothelial endoplasmic reticulum contains both inositol tris-phosphate receptors and ryanodine receptors but that the former class are more densely distributed.

Abstract A new method of measuring cytoplasmic free Ca 2+ ([Ca 2+ ] i ) of individual intact cardiovascular endothelial cells by using imaging fluorescence microscopy was designed. Application of agonist to the aortic or pulmonary valve of the rabbit triggered an increase in [Ca 2+ ] i , which depended on the existence of endothelium on the surface of the valve. Under resting conditions, sudden reversal of the Na + gradient by substituting external Na + with N -methyl d -glucamine (NMDG) resulted in a [Ca 2+ ] i spike, which then returned toward the resting level. Increasing intracellular Na + concentration ([Na + ] i ) by application of ouabain or monensin induced a sustained [Ca 2+ ] i increase. Na + substitution by NMDG during the agonist- or monensin-induced [Ca 2+ ] i increase gave rise to a further [Ca 2+ ] i spike, which subsequently declined to a level higher than that before removal of external Na + . A selective inhibitor of Na + -Ca 2+ exchange, 3′,4′-dichlorobenzamyl (DCB), abolished the transient [Ca 2+ ] i increase induced by Na + substitution, and Mg 2+ , an inorganic inhibitor of Na + -Ca 2+ exchanger, markedly reduced this transient [Ca 2+ ] i increase. On the other hand, the selective Na + -H + exchanger blocker 5-( N,N -hexamethylene)amiloride (HMA) did not abolish the transient [Ca 2+ ] i increase caused by Na + substitution. In summary, decreasing the Na + gradient of the endothelial cells through either receptor stimulation (agonist), Na + -K + pump inhibition (ouabain), pretreatment with Na + ionophore (monensin), or reversing the Na + gradient through Na + substitution (NMDG) all increased [Ca 2+ ] i . This raised [Ca 2+ ] i was antagonized by agents such as DCB or Mg 2+ , which are thought to inhibit Na + -Ca 2+ exchange, but not by HMA, an inhibitor of Na + -H + exchange. Taken together, these results strongly imply the presence of Na + -Ca 2+ exchange as a viable mechanism for Ca 2+ transport in intact cardiovascular endothelium and that the Ca 2+ entry component is enhanced when [Na + ] i is elevated.

We address the importance of cytoplasmic nanospaces in Ca(2+) transport and signalling in smooth muscle cells and how quantitative modelling can shed significant light on the understanding of signalling mechanisms. Increasingly more convincing evidence supports the view that these nanospaces--nanometre-scale spaces between organellar membranes, hosting cell signalling machinery--are key to Ca(2+) signalling as much as Ca(2+) transporters and Ca(2+) storing organelles. Our research suggests that the origin of certain diseases is to be sought in the disruption of the proper functioning of cytoplasmic nanospaces. We begin with a historical perspective on the study of smooth muscle cell plasma membrane-sarcoplasmic reticulum nanospaces, including experimental evidence of their role in the generation of asynchronous Ca(2+) waves. We then summarize how stochastic modelling approaches have aided and guided our understanding of two basic functional steps leading to healthy smooth muscle cell contraction. We furthermore outline how more sophisticated and realistic quantitative stochastic modelling is now being employed not only to deepen our understanding but also to aid in the hypothesis generation for further experimental investigation.

Exposure to particulate matter air pollution may be an independent risk factor for cardiovascular morbidity and mortality; however, the biological mechanisms are unclear. We hypothesize that exposure to diesel exhaust (DE), an important source of traffic-related particulate air pollution, promotes changes of atherosclerotic plaque component that may lead to plaque vulnerability.30-week old ApoE knockout mice fed with regular chow inhaled DE (at 200 μg/m(3) of particulate) or filtered-air (control) for 7 weeks (6 h/day, 5 days/week) (12 mice/group). Total number of alveolar macrophages (p0.01) and alveolar macrophages positive for particles (p0.0001) were more than 8-fold higher after DE inhalation than the control. DE inhalation caused 1.5 to 3-fold increases in plaque lipid content (p0.02), cellularity (p0.02), foam cell formation (p0.04), and smooth muscle cell content (p0.05). The expression of oxidative stress markers, iNOS, CD36, and nitrotyrosine was significantly increased by 1.5 to 2-fold in plaques, with enhanced systemic lipid and DNA oxidation (p0.02). Increased foam cells and the expression of iNOS (R(2)=0.72, p=0.0081) and CD36 (R(2)=0.49, p=0.015) in plaques were positively correlated with the magnitude of DE exposure.Exposure to DE promotes changes in atherosclerotic plaques characteristic of unstable vulnerable plaques. Increased systemic and plaque oxidative stress markers suggest that these changes in plaques could be due to DE-induced oxidative stress.

Background: Vascular smooth muscle cell migration and accumulation in response to growth factors extensively contribute to the development of intimal thickening within the vessel wall. Cumulative evidence has shown that actin cytoskeleton polymerization and rearrangement are critical steps during cellular spreading and migration. Integrin-linked kinase, an intracellular serine/threonine kinase, is a cytoplasmic interactor of integrin beta-1 and beta-3 receptors regulating cell-cell and/or cell-extracellular matrix interaction, cell contraction, extracellular matrix modification, and cell spreading and migration in response to various stimuli. However, the regulatory role of ILK during vascular smooth muscle cell migration and the importance of integrin signaling in occlusive vascular diseases are not yet fully elucidated. Results In the present study, we report that integrin-linked kinase controls mouse aortic smooth muscle cell migration in response to platelet-derived growth factor. We have also identified p38 mitogen activated protein kinase as a downstream signaling pathway of the integrin-linked kinase that regulates platelet-derived growth factor-induced actin polymerization and smooth muscle cell migration. Conclusion This study will provide new insights into the potential therapeutic value of modulating integrin signaling in an attempt to block or delay smooth muscle cell migration and the progression of vascular diseases.

The mechanism of aluminumfloride (AlF)-induced Ca2+ sensitization was explored in alpha-toxin-permeabilized rabbit mesenteric artery. In the presence of 0.18 microM Ca2+ and deferoxamine, a strong chelator of aluminum (Al3+), fluoride (F-; applied in the form of NaF) induced very slow tension development, while in the presence of tracer levels of Al3+ tension developed rapidly possibly due to formation of Al-F complexes (especially AlF4-). As a result, AlF significantly shifted the relationship between tension development and free Ca2+ concentration in the Ca(2+)-EGTA buffer (pCa-tension curve) to the left. The rate of the tension development also depended on the EGTA concentration: increasing the EGTA concentration from 0.5 to 10 mM markedly decreased the maximal rate of contraction ((dT/dt)max), probably due to chelation of Al3+ by EGTA, without effect on the maximal tension (delta Tmax). The AlF-induced Ca2+ sensitization could be reversed by extensive washing with relaxing solution (pCa greater than 8), in contrast to the contractions induced by guanosine 5'-[gamma-thio]triphosphate (GTP gamma s; a non-hydrolyzable GTP analogue) or phorbol 12,13-dibutyrate (PDBu) which were irreversible. However, the action of all the compounds appeared to be mediated through a H-7 (1-[5-isoquinolinesulfonyl]-2-methylpiperazine dihydrochloride)-sensitive pathway, and no additive effects among them were observed. In addition, GDP increased (dT/dt)max due to AlF without changing delta Tmax, whereas guanosine 5'-[beta-thio]diphosphate (GDP beta s; a non-hydolyzable GDP analogue) decreased both parameters. These findings suggest that AlF acts on G-proteins to enhance Ca2+ sensitivity of contractile elements through a H-7-sensitive pathway.

During development of thoracic aortic aneurysms in a mouse model of Marfan syndrome, upregulation of matrix metalloproteinase (MMP)-2 and -9 was accompanied by compromised aortic constriction and endothelium-dependent relaxation. Losartan has been proposed for the prevention of thoracic aortic aneurysm. We hypothesized that losartan would suppress MMP-2/-9 activation and improve aortic vasomotor function in this model.A well-characterized mouse model of Marfan syndrome (Fbn1(C1039G/+)) was used. Starting at 6 weeks old, Marfan mice were untreated or given losartan (0.6 g.L(-1) in drinking water, n= 30). The littermate Fbn1(+/+) mice served as control. Thoracic aortas were studied at 3, 6 and 9 months by histology and by contractility assays in isolated segments in vitro.Losartan improved elastic fibre organization and increased aortic breaking stress. Losartan reduced the activity and protein expression of MMP-2 and MMP-9 at all ages. Aortic constriction in response to membrane depolarization or phenylephrine was increased by losartan at 3 and 9 months by 100-200%. Active force of aortic smooth muscle was also increased at 6 and 9 months. Acetylcholine-induced endothelium-dependent relaxation was improved by 30% after 3 months of losartan treatment, but such improvement disappeared with longer duration of treatment, accompanied by reduced phosphorylation of endothelial nitric oxide (NO) synthase(Ser1177), Akt(Thr308) and Akt(Ser473), compared with the control.Losartan improved the contractile function of aorta and reduced MMP activation. However, the endothelial NO pathway remained suppressed in the thoracic aorta during losartan treatment, which might limit its long-term benefits in Marfan syndrome.

We present a stochastic computational model to study the mechanism of signalling between a source and a target ionic transporter, both localized on the plasma membrane (PM) and in intracellular nanometre-scale subplasmalemmal signalling compartments comprising the PM, the sarcoplasmic reticulum (SR), Ca2+ and Na+ transporters, and the intervening cytosol. We refer to these compartments, sometimes called junctions, as cytoplasmic nanospaces or nanodomains. In the chain of events leading to Ca2+ influx for SR reloading during asynchronous Ca2+ waves in vascular smooth muscle (VSM), the physical and functional link between non-selective cation channels (NSCC) and Na+/Ca2+ exchangers (NCX) needs to be elucidated in view of two recent findings: the identification of the transient receptor potential canonical channel 6 (TRPC6) as a crucial NSCC in VSM cells and the observation of localized cytosolic [Na+] transients following purinergic stimulation of these cells. Having previously helped clarify the Ca2+ signalling step between NCX and SERCA behind SR Ca2+ refilling, this quantitative approach now allows us to model the upstream linkage of NSCC and NCX. We have implemented a random walk (RW) Monte Carlo (MC) model with simulations mimicking a Na+ diffusion process originating at the NSCC within PM-SR junctions. The model calculates the average [Na+] in the nanospace and also produces [Na+] profiles as a function of distance from the Na+ source. Our results highlight the necessity of a strategic juxtaposition of the relevant signalling channels as well as other physical structures within the nanospaces to permit adequate [Na+] build-up to provoke NCX reversal and Ca2+ influx to refill the SR., 16 pages, 9 figures; an abridged version submitted for publication

In many vascular smooth muscle cells, physiological and pharmacological agonists initiate oscillatory fluctuations in intracellular Ca(2+) to initiate and maintain vasoconstriction. These oscillations are supported by the underlying cellular ultrastructure, particularly the close apposition between the plasma membrane (PM) and superficial sarcoplasmic reticulum (SR), the so-called PM-SR junctions, which are important for SR Ca(2+) refilling. We hypothesize that the disappearance of PM-SR junctions during aging and/or disease is directly related to the disappearance of agonist-induced Ca(2+) oscillations. We compared phenylephrine-mediated Ca(2+) signals and contraction in human and murine smooth muscle cells in small mesenteric arteries and also employed electron microscopy to examine the cytoplasmic distribution of the SR. Phenylephrine elicited tonic contractions in both types of vessels, asynchronous Ca(2+) oscillations in the mouse mesenteric smooth muscle cells, but only single transient Ca(2+) signals in the human mesenteric smooth muscle cells. While nifedipine inhibited 90% of the phenylephrine-induced tonic contraction in mouse mesenteric arteries, it only slightly attenuated tonic contraction in human mesenteric arteries, although the nifedipine-resistant component was abolished by the Rho-kinase blocker 1-(5-Isoquinolinylsulfonyl)homopiperazine dihydrochloride (HA-1077). Furthermore, superficial SR was found to be abundant in the mouse vessels and many PM-SR junctions were observed, but the smooth muscle of human mesenteric arteries had far less peripheral SR and was almost devoid of PM-SR junctions. As PM-SR junctions are essential for the maintenance of Ca(2+) oscillations, the change in Ca(2+) signalling pattern in the relatively old human patients was due to impaired SR refilling.

We have described that the progression of thoracic aortic aneurysm in Marfan syndrome is accompanied with aortic vascular dysfunction. In the present study, we hypothesized that the impaired contractile function and endothelial-dependent relaxation could be resulted from oxidative stress in the thoracic aorta. Adrenergic contraction and cholinergic relaxation of thoracic aortae from mice (n = 40; age = 3, 6, 9 months) heterozygous for FBN1 allele (Fbn1C1039G/+), a well-defined model of Marfan syndrome, were compared with those from control (n = 40). The aortic 8-isoprostane level, an oxidative stress marker, was 32–50% greater in the Marfan group than in the control. Pre-incubation with superoxide dismutase (SOD) improved the phenylephrine-induced contraction and the sensitivity to acetylcholine in Marfan aortae, but not in controls. The phenylephrine-contraction in Marfan aortae was potentiated by 1400 W, an inducible nitric oxide synthase (iNOS) inhibitor, and allopurinol, a xanthine oxidase inhibitor. Acetylcholine-induced relaxation was restored by apocynin, an inhibitor of NAD(P)H oxidase. Protein expression of SOD-1 and SOD-2 was decreased in Marfan aortae, whereas that of xanthine oxidase, iNOS, and the enzymatic subunits of NAD(P)H oxidase was increased. The vasomotor dysfunction in Marfan thoracic aortae could be associated with accumulation of oxidative stress due to unbalanced protein expression of superoxide-producing and superoxide-eliminating enzymes.

Large-conductance Ca2(+)-activated K+ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 microM), an agonist which releases Ca2+ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 microM), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca2+ concentration at the cytoplasmic side above 10(-7) mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (Ki) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca2+ sensitivity of the channel, shifting the Ca2+ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca2(+)-activated K+ channel in airway smooth muscle. This ATP modulation of Ca2(+)-activated K+ channels might serve as an important mechanism linking energy status and the contractile state of the cells.

Following the recent observation of localized cytosolic subplasmalemmal [Na+] elevations (LNats) in rat aortic smooth muscle cells, we discuss here the current evidence for the structural and molecular roles of cytosolic nanodomains at close junctions of the plasma membrane (PM) and sarcoplasmic reticulum (SR) in the generation of LNats. These junctions, the loss of which might contribute to vascular aging and disease, provide a platform for ion metabolism signalplexes and the interaction of localized Na+ and Ca2+ gradients. We moreover suggest the existence in the junctions of a Na+ diffusional barrier as a necessary condition for the generation of LNats. LNats are likely a fundamental feature of near membrane ion signaling in many cell types, and their discovery offers new possibilities for elucidating the mechanism, function and pathogenesis of Na+ and Ca2+ signaling nanodomains.

The changes in isometric force and the underlying fluctuations in intracellular free calcium concentration ([Ca2+]i) were monitored simultaneously in thin sheets of rabbit inferior vena cava loaded with the fluorescent Ca2+ indicator fura-2. In resting tissues bathed in physiological saline solution, the estimated [Ca2+]i was approximately 105 nM. The alpha-adrenergic agonist norepinephrine (10 microM) caused an initial rise in [Ca2+]i to 264 nM during force development, which dropped to 216 nM during force maintenance. The maintained norepinephrine-induced increase in force and [Ca2+]i was reversed in Ca2(+)-free (2 mM EGTA) solution. Membrane depolarization by high K+ (80 mM) significantly increased [Ca2+]i to 234 nM. Compared to norepinephrine, high K+ caused about the same steady-state increase in [Ca2+]i, but a smaller increase in force. [Ca2+]i/force curves were constructed at different concentrations of extracellular Ca2+, with either norepinephrine or high K+ as a stimulant. The curve generated with norepinephrine was located to the left of that generated with high K+.

The Na+/Ca2+ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca2+ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca2+ influx by the NCX (1) is the primary mechanism of Ca2+ entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na+ indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na+] ([Na+]i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na+]i elevations and localized [Na+]i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 micromol/L) and 2-amino-4-phosphonobutyrate (75 micromol/L) reduced both global and localized [Na+]i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca2+ entry with KB-R7943 (10 micromol/L) enhanced the LNats, whereas the global cytosolic [Na+] signal was unaffected. Inhibition of mitochondrial Na+ uptake with CGP-37157 (10 micromol/L) increased both LNats and global cytosolic [Na+] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca2+ entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6-containing channels.

Thoracic aortic aneurysm (TAA) is the life-threatening complication of Marfan syndrome (MFS), a connective tissue disorder caused by mutations in the fibrillin-1 gene. TAA is characterized by degradation of elastic fiber, suggesting the involvement of matrix metalloproteinase (MMP)-2 and -9, the activation of which is regulated by TIMP (tissue inhibitor of MMP) types 1 and 2. We hypothesized that MMP-2 and -9 were upregulated during TAA formation in Marfan syndrome, causing loss of elastic fibers and structural integrity. We studied mice, from 3 to 12 months, heterozygous for a mutant Fbn 1 allele encoding a cysteine substitution in fibrillin-1 ( Fbn1 C1039G/+ , designated as “Marfan” mice) (n=120), the most common class of mutation in Marfan syndrome. The littermates, Fbn1 +/+ served as controls (n=120). In Marfan aneurysmal thoracic aorta, mRNA and protein expression of MMP-2 and -9 were detected at 3 months and peaked at 6 months of age, accompanied by severe elastic fiber fragmentation and degradation. From 3 to 9 months, the MMP-2/TIMP-2 ratio increased by 43% to 63% compared with the controls. Dilated thoracic aorta demonstrated increased elasticity but distention caused a pronounced loss of contraction, suggesting weakening of the aortic wall. Breaking stress of the aneurysmal aorta was 70% of the controls. Contraction in response to depolarization and receptor stimulation decreased in the aneurysmal thoracic aorta by 50% to 80%, but the expression of α-smooth muscle actin between the 2 strains was not significantly different. This report demonstrates the upregulation of MMP-2 and -9 during TAA formation in Marfan syndrome. The resulting elastic fiber degeneration with deterioration of the aortic contraction and mechanical properties may explain the pathogenesis of TAA.

Diabetes mellitus impairs endothelial function, which can be considered as the hallmark in the development of cardiovascular diseases. Hyperglycemia, hyperinsulinemia, and hyperlipidemia are believed to contribute to endothelial dysfunction. In the present study, we investigated the possible links among these plasma metabolic markers and endothelial function in a mouse model during the development of type 2 diabetes. C57BL/6J-Lepob/ob mice at 8, 12, and 16 weeks were used to study endothelial function during the establishment of type 2 diabetes. Endothelial function was accessed in vitro in the thoracic aorta by measuring acetylcholine (ACh)-stimulated vasodilatation. Blood plasma was obtained for the measurements of glucose, insulin, triglycerides, and cholesterol levels. Correlation and multiple regression analysis revealed strong negative associations between the ACh responsiveness and the plasma levels of glucose, insulin, and lipid profiles at the age of 8 weeks. Associations were observed at neither older age nor in C57BL/6J mice. In conclusion, the increase in plasma levels of glucose, insulin, and lipids is associated with the impairment of the endothelial function during the early stage of the development of type 2 diabetes. The loss of correlation at an older age suggests multifactorial regulation of endothelial function and cardiovascular complications at later stages of the disease.

Reversal of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) has been shown to mediate Ca(2+) influx in response to activation of G-protein linked receptors. Functional coupling of reverse-mode NCX with canonical transient receptor potential channels (TRPC), specifically TRPC6, has recently been demonstrated by our laboratory to mediate Ca(2+) influx in rat aortic smooth muscle cells (RASMCs) following ATP stimulation. In this communication, we provide further detail of this functional coupling by indirectly measuring NCX reversal. We found that NCX reversal, induced by the removal of extracellular Na(+), was increased following stimulation with ATP and the diacylglycerol analog 1-Oleoyl-2-acetyl-sn-glycerol. This increased NCX reversal was attenuated by SKF-96365, an inhibitor of non-selective cation channels, and by activation of protein kinase C with phorbol ester 12-tetradecanoylphorbol-13 acetate. These data are consistent with the known properties of TRPC6 and further support that functional coupling of TRPC6 and NCX occurs via a receptor-operated, rather than store-operated, cascade in RASMCs.