To provide insight into mitochondrial function in vivo, we evaluated the 3D spatial relationship between capillaries, mitochondria, and muscle fibers in live mice.
3D volumes of in vivo murine Tibialis anterior muscles were imaged by multi-photon microscopy (MPM). Muscle fiber type, mitochondrial distribution, number of capillaries, and capillary-to-fiber contact were assessed. The role of myoglobin-facilitated diffusion was examined in myoglobin knockout mice. Distribution of GLUT4 was also evaluated in the context of the capillary and mitochondrial network.
MPM revealed that 43.6 ± 3.3% of oxidative fiber capillaries had ≥ 50% of their circumference embedded in a groove in the sarcolemma, in vivo. Embedded capillaries were tightly associated with dense mitochondrial populations lateral to capillary grooves and nearly absent below the groove. Mitochondrial distribution, number of embedded capillaries, and capillary-to-fiber contact were proportional to fiber oxidative capacity and unaffected by myoglobin knockout. GLUT4 did not preferentially localize to embedded capillaries.
Embedding capillaries in the sarcolemma may provide a regulatory mechanism to optimize delivery of oxygen to heterogeneous groups of muscle fibers. We hypothesize that mitochondria locate to paravascular regions due to myofibril voids created by embedded capillaries, not to enhance the delivery of oxygen to the mitochondria.
Two-photon microscopy; 3D quantitative imaging; muscle mitochondria; myoglobin; vasculature; microcirculation; GLUT4
To create accurate, high resolution 3D reconstructions of neovasculature structures in xenografted tumors and Matrigel plugs for quantitative analyses in angiogenesis studies in animal models.
The competent neovasculature within xenografted solid tumors or Matrigel plugs in mice was perfused with Microfil, a radio-opaque, hydrophilic polymerizing contrast agent, by systemic perfusion of the blood circulation via the heart. The perfused tumors and plugs were resected and scanned by X-ray micro-computed tomography to generate stacks of 2D images showing the radio-opaque material. A non-biased, precise post-processing scheme was employed to eliminate background X-ray absorbance from the extra-vascular tissue. The revised binary image stacks were compiled to reveal the Microfil-casted neovasculature as 3D reconstructions. Vascular structural parameters were calculated from the refined 3D reconstructions using the scanner software.
Clarified 3D reconstructions were sufficiently precise to allow measurements of vascular architecture to a diametric limit of resolution of 3 μm in tumors and plugs.
Ex vivo micro-computed tomography can be used for 3D reconstruction and quantitative analysis of neovasculature including microcirculation in solid tumors and Matrigel plugs. This method can be generally applied for reconstructing and measuring vascular structures in 3 dimensions.
Micro-computed tomography (microCT); Vascular imaging; Tumor angiogenesis; Matrigel plug; Microvasculature
Fenestrations are pores in the liver sinusoidal endothelium that facilitate the transfer of particulate substrates between the sinusoidal lumen and hepatocytes. Fenestrations express caveolin-1 and have structural similarities to caveolae, therefore might be a form of caveolae and caveolin-1 may be integral to fenestration structure and function. Therefore, fenestrations were studied in the livers of caveolin-1 knockout mice.
Scanning, transmission and immunogold electron microscopic techniques were used to study the liver sinusoidal endothelium and other tissues in caveolin-1 knockout and wild-type mice.
Comparison of fenestrations in wild-type and knockout mice did not reveal any differences on either scanning or transmission electron microscopy. The diameter of the fenestrations was not significantly different (74 ± 13 nm knockout mice vs 78 ± 12 nm wild-type mice) nor was the fenestration porosity (6.5 ± 2.1 knockout vs 7.3 ± 2.4% wild-type mice). In contrast, adipocytes and blood vessels in other tissues lacked caveolae in the knockout mice. Caveolin-1 immunogold of livers of wild-type mice indicated sparse expression in sinusoidal endothelial cells.
The normal structure of fenestrations in the liver sinusoidal endothelium is not dependent upon caveolin-1 and fenestrations are not a form of caveolae.
fenestrations; fenestrae; sinusoidal endothelial cell; liver; hepatic; caveolin-1; knockout mouse; electron microscopy; immunogold
In the adult, angiogenesis leads to an expanded microvascular network as new vessel segments are added to an existing microcirculation. Necessarily, growing neovessels must navigate through tissue stroma as they locate and grow toward other vessel elements. We have a growing body of evidence demonstrating that angiogenic neovessels reciprocally interact with the interstitial matrix of the stroma resulting in directed neovascular growth during angiogenesis. Given the compliance and the viscoelastic properties of collagen, neovessel guidance by the stroma is likely due to compressive strain transverse to the direction of primary tensile forces present during active tissue deformation. Similar stromal strains control the final network topology of the new microcirculation, including the distribution of arterioles, capillaries, and venules. In this case, stromal-derived stimuli must be present during the post-angiogenesis remodeling and maturation phases of neovascularization to have this effect. Interestingly, the preexisting organization of vessels prior to the start of angiogenesis has no lasting influence on the final, new network architecture. Combined, the evidence describes interplay between angiogenic neovessels and stroma that is important in directed neovessel growth and invasion. This dynamic is also likely a mechanism by which global tissue forces influence vascular form and function.
angiogenesis; stroma; matrix; neovessel; remodeling
The formation of the cardiovasculature, consisting of both the heart and blood vessels, is a critical step in embryonic development and relies on three processes termed vasculogenesis, angiogenesis, and vascular remodeling. The transmembrane protein NRP1 is an essential modulator of embryonic angiogenesis with additional roles in vessel remodeling and arteriogenesis. NRP1 also enhances arteriogenesis in adults to alleviate pathological tissue ischemia. However, in certain circumstances, vascular NRP1 signaling can be detrimental, as it may promote cancer by enhancing tumor angiogenesis or contribute to tissue edema by increasing vascular permeability. Understanding the mechanisms of NRP1 signaling is, therefore, of profound importance for the design of therapies aiming to control vascular functions. Previous work has shown that vascular NRP1 can variably serve as a receptor for two secreted glycoproteins, the VEGF-A and SEMA3A, but it also has a poorly understood role as an adhesion receptor. Here, we review current knowledge of NRP1 function during blood vessel growth and homeostasis, with special emphasis on the vascular roles of its multiple ligands and signaling partners.
angiogenesis; VEGF; VEGF-A; SEMA3A; neuropilin; NRP1; VEGFR2; endothelial cells; angiogenesis; arteriogenesis; vascular permeability
Arterioles, capillaries and venules all actively change their cellular functions and phenotypes during inflammation in ways that are essential for maintenance of homeostasis and self-defense, and are also associated with many inflammatory disorders. Endothelial cells (ECs), together with pericytes and extracellular matrix proteins, can regulate blood flow, the coagulation cascade, fluid and solute exchange, and leukocyte trafficking. While capillary and venular functions in inflammation are well characterized, the arteriolar contribution to inflammation has only recently come into focus. Arterioles differ from venules in structure, EC morphology, shear environment, expression and distribution of surface ligands, hence regulation and function of arteriolar wall cells during inflammation may also be distinct from venules. Recent work indicates that in response to pro-inflammatory stimuli, arterioles alter barrier function, and support leukocyte and platelet interactions through upregulation of adhesion molecules. This suggests that in addition to their role in blood flow regulation, arterioles may also participate in inflammatory responses. In this review we will discuss mechanisms that characterize arteriolar responses to proinflammatory stimuli. We will detail how distinct arteriolar features contribute to regulation of barrier function and leukocyte-EC interactions in inflammation, and further highlight the potential priming effects of arteriolar responses on venular function and progression of inflammatory responses.
endothelial cells; morphology; permeability; leukocytes; neutrophils; adhesion molecules; shear stress; inflammatory responses
Hemodynamic properties of vascular beds are of great interest in a variety of clinical and laboratory settings. However, there presently exists no automated, accurate, technically simple method for generating blood velocity maps of complex microvessel networks. Here we present a novel algorithm that addresses this problem by applying pixel-by-pixel cross-correlation to video data. Temporal signals at every spatial coordinate are compared with signals at neighboring points, generating a series of correlation maps from which speed and direction are calculated. User assisted definition of vessel geometries is not required, and sequential data are analyzed automatically, without user bias. Velocity measurements are validated against the dual-slit method and against capillary flow with known velocities. The algorithm is tested in three different biological models. Along with simultaneously acquired hemoglobin saturation and vascular geometry information, the hemodynamic maps presented here demonstrate an accurate, quantitative method of analyzing dynamic vascular systems.
We compare Reconstructed Microvascular Networks (RMN) to Parallel Capillary Arrays (PCA) under several simulated physiological conditions to determine how the use of different vascular geometry affects oxygen transport solutions.
Three discrete networks were reconstructed from intravital video microscopy of rat skeletal muscle (84×168×342 μm, 70×157×268 μm and 65×240×571 μm) and hemodynamic measurements were made in individual capillaries. PCAs were created based on statistical measurements from RMNs. Blood flow and O2 transport models were applied and the resulting solutions for RMN and PCA models were compared under 4 conditions (rest, exercise, ischemia and hypoxia).
Predicted tissue PO2 was consistently lower in all RMN simulations compared to the paired PCA. PO2 for 3D reconstructions at rest were 28.2±4.8, 28.1±3.5, and 33.0±4.5 mmHg for networks I, II, and III compared to the PCA mean values of 31.2±4.5, 30.6±3.4, and 33.8±4.6 mmHg. Simulated exercise yielded mean tissue PO2 in the RMN of 10.1±5.4, 12.6±5.7, and 19.7±5.7 mmHg compared to 15.3±7.3, 18.8±5.3, and 21.7±6.0 in PCA.
These findings suggest that volume matched PCA yield different results compared to reconstructed microvascular geometries when applied to O2 transport modeling; the predominant characteristic of this difference being an over estimate of mean tissue PO2. Despite this limitation, PCA models remain important for theoretical studies as they produce PO2 distributions with similar shape and parameter dependence as RMN.
blood flow; parallel capillary networks; oxygen transport modeling; computational model; three-dimensional microvascular reconstruction
To use the obese Zucker rat (OZR) model of the metabolic syndrome to determine the impact of dilator stimuli on myogenic activation (MA) of gracilis arterioles (GA) and middle cerebral arteries (MCA). We tested the hypothesis that increased oxidant stress and thromboxane A2 (TxA2) exacerbate MA, and prevent its blunting with dilator stimuli, in OZR.
GA/MCA from OZR and lean Zucker rats (LZR) were pressurized ex vivo. MA was determined under control conditions and following challenge with acetylcholine, hypoxia and adenosine. Responses were also evaluated after pre-treatment with TEMPOL (antioxidant) and SQ-29548 (PGH2/TxA2 receptor antagonist).
MA was increased (and dilator responses decreased) in GA/MCA from OZR, dependent on the endothelium and ROS. In GA, the impact of ROS on MA and dilator effects was largely via TxA2, while in MCA, this appeared was more dependent on NO bioavailability. Intrinsic responses of GA/MCA to carbacyclin, U46619, and NO donors were similar between strains.
A developing ROS-based endothelial dysfunction in MCA and GA of OZR contributes to an enhanced MA of these vessels. While treatment of GA/MCA with TEMPOL attenuates MA in OZR, the mechanistic contributors to altered MA, distal to ROS, differ between the two resistance vessels.
regulation of vascular tone; rodent models of metabolic syndrome; peripheral resistance; microcirculation
Obese subjects exhibit decreased exercise capacity (VO2max). We have shown that vascular KATP channel mediates arteriolar dilation to muscle contraction. We hypothesize that exercise capacity is decreased in obesity due to impaired vascular KATP function.
VO2max was measured in LZR and OZR by treadmill running before and following treatment with the KATP blocker glibenclamide i.p. One week later the spinotrapezius muscle was prepared for in vivo microscopy. Arcade arteriolar diameters were measured following muscle contraction or application of the KATP opener cromakalim before and after glibenclamide application. In additional animals, LZR and OZR were treated with apocynin for 5 weeks. VO2max and arteriolar dilation experiments were repeated.
OZR exhibited decreased VO2max, functional and cromakalim-induced vasodilation as compared to LZR. Glibenclamide had no effect on VO2max and functional vasodilation in OZR but significantly inhibited responses in LZR. Vascular superoxide levels and NADPH oxidase activity were increased in OZR but reduced in apocynin-treated OZR. Apocynin increased the VO2max, functional and cromakalim-induced vasodilation in OZR with no effect in LZR.
Exercise capacity is dependent on vascular KATP channel function. The reduced exercise capacity in OZR appears to be due in part to superoxide-mediated impairment in vascular KATP function.
Obese; vascular; NADPH oxidase; superoxide; KATP channels
The classical model of metabolic regulation of blood flow in muscle tissue implies the maintenance of basal tone in arterioles of resting muscle and their dilation in response to exercise and/or tissue hypoxia via the evoked production of vasodilator metabolites by myocytes. A century-long effort to identify specific metabolites responsible for explaining active and reactive hyperemia has not been successful. Furthermore, the metabolic theory is not compatible with new knowledge on the role of physiological radicals (e.g., nitric oxide, NO, and superoxide anion, O2−) in the regulation of microvascular tone. We propose a model of regulation in which muscle contraction and active hyperemia are considered the physiologically normal state. We employ the “bang-bang” or “on/off” regulatory model which makes use of a threshold and hysteresis; a float valve to control the water level in a tank is a common example of this type of regulation. Active bang-bang regulation comes into effect when the supply of oxygen and glucose exceeds the demand, leading to activation of membrane NADPH oxidase, release of O2− into the interstitial space and subsequent neutralization of the interstitial NO. Switching arterioles on/off when local blood flow crosses the threshold is realized by a local cell circuit with the properties of a bang-bang controller, determined by its threshold, hysteresis and dead-band. This model provides a clear and unambiguous interpretation of the mechanism to balance tissue demand with a sufficient supply of nutrients and oxygen.
metabolic regulation; nitric oxide; on/off regulation; oxygen; superoxide anion
We sought to test the hypothesis that turmeric-derived curcuminoids limit reperfusion brain injury in an experimental model of stroke via blockade of early microvascular inflammation during reperfusion.
Male Sprague Dawley rats subjected to middle cerebral artery occlusion and reperfusion (MCAO/R) were treated with turmeric-derived curcuminoids (vs. vehicle) 1 hour prior to reperfusion (300 mg/kg ip). Neutrophil adhesion to the cerebral microcirculation and measures of neutrophil and endothelial activation were assayed during early reperfusion (0–4 hours); cerebral infarct size, edema and neurological function were assessed at 24 h. Curcuminoid effects on TNFα−stimulated human brain microvascular endothelial cell (HBMVEC) were assessed.
Early during reperfusion following MCAO, curcuminoid treatment decreased neutrophil rolling and adhesion to the cerebrovascular endothelium by 76% and 67% and prevented >50% of the fall in shear rate. The increased number and activation state (CD11b and ROS) of neutrophils were unchanged by curcuminoid treatment, while increased cerebral expression of TNFα and ICAM-1, a marker of endothelial activation, were blocked by >30%. Curcuminoids inhibited NF-κB activation and subsequent ICAM-1 gene expression in HBMVEC.
Turmeric derived curcuminoids limit reperfusion injury in stroke by preventing neutrophil adhesion to the cerebrovascular microcirculation and improving shear rate by targeting the endothelium.
ischemia; reperfusion; stroke; curcuminoids; neutrophil; endothelium
We previously showed that acute alcohol intoxication (AAI) reduces lymphatic myogenic constriction in response to step increases in luminal pressure. Because of the known role of Ca2+ in smooth muscle contractile responses, we investigated how alcohol impacts cyclic Ca2+ and whether changes in RhoA/ROCK mediated Ca2+ sensitivity underlie the alcohol-induced reduction of myogenic responsiveness.
AAI was produced by intragastric administration of 30% alcohol in rats. Mesenteric lymphatics were cannulated and loaded with Fura-2 AM to [Ca2+]i for 30 min after AAI. Active GTP-bound RhoA levels were determined by ELISA. To determine ROCK's ability to restore myogenic responsiveness following AAI, isolated lymphatics were transfected with constitutively active ca-ROCK protein.
Lymphatics from alcohol-treated rats displayed significantly larger Ca2+ transients. Also, step increases in luminal pressure caused a gradual rise in the basal [Ca2+]i between transients that was greater in lymphatics submitted to AAI, compared to vehicle control. RhoA-GTP was significantly reduced in lymphatics from the AAI group, compared to vehicle control. Transfection with ca-ROCK protein restored the myogenic response of lymphatic vessels isolated from AAI animals.
The data strongly suggest that the alcohol-induced inhibition of mesenteric lymphatic myogenic constriction is mediated by reduced RhoA/ROCK-mediated Ca2+ sensitivity.
calcium; lymphatic smooth muscle; myogenic tone; ROCK; ethanol
Lymphatic filariasis, one of the most debilitating diseases associated with the lymphatic system, affects over a hundred million people worldwide and manifests itself in a variety of severe clinical pathologies. The filarial parasites specifically target the lymphatics and impair lymph flow, which is critical for the normal functions of the lymphatic system in maintenance of body fluid balance and physiological interstitial fluid transport. The resultant contractile dysfunction of the lymphatics causes fluid accumulation and lymphedema, one of the major pathologies associated with filarial infection. In this review, we take a closer look at the contractile mechanisms of the lymphatics, its altered functions and remodeling during an inflammatory state and how it relates to the severe pathogenesis underlying a filarial infection. We further elaborate on the complex host parasite interactions, and molecular mechanisms contributing to the disease pathogenesis. The overall emphasis is on elucidating some of the emerging concepts and new directions that aim to harness the process of lymphangiogenesis or enhance contractility in a dysfunctional lymphatics, thereby restoring the fluid imbalance and mitigating the pathological conditions of lymphatic filariasis.
The risk for cardiovascular disease increases with advancing age; however, the chronological development of heart disease differs in males and females. The purpose of this study was to determine whether age-induced alterations in responses of coronary arterioles to the endogenous vasoconstrictor, endothelin, are sex-specific. Coronary arterioles were isolated from young and old male and female rats to assess vasoconstrictor responses to endothelin (ET), and ETa and ETb receptor inhibitors were used to assess receptor-specific signaling. In intact arterioles from males, ET-induced vasoconstriction was reduced with age, whereas age increased vasoconstrictor responses to ET in intact arterioles from female rats. In intact arterioles from both sexes, blockade of either ETa or ETb eliminated age-related differences in responses to ET; however, denudation of arterioles from both sexes revealed age-related differences in ETa-mediated vasoconstriction. In arterioles from male rats, ETa receptor protein decreased, whereas ETb receptor protein increased with age. In coronary arterioles from females, neither ETa nor ETb receptor protein changed with age, suggesting age-related changes in ET signaling occur downstream of ET receptors. Thus, aging-induced alterations in responsiveness of the coronary resistance vasculature to endothelin are sex-specific, possibly contributing to sexual dimorphism in the risk of cardiovascular disease with advancing age.
rat; vasodilation; BQ123; BQ788
The acute implantation of a cranial window for studying cerebroarteriolar reactivity in living animals involves a highly surgically-invasive craniotomy procedure at the time of experimentation, which limits its application in severely ill animals such as in the experimental murine model of cerebral malaria (ECM). To overcome this problem, a chronic window implantation scheme was designed and implemented.
A partial craniotomy is first performed by creating a skull bone flap in the healthy mice, which are then left to recover for 1–2 weeks, followed by infection to induce ECM. Uninfected animals are utilized as control. When cranial superfusion is needed, the bone flap is retracted and window implantation completed by assembling a perfusion chamber for compound delivery to the exposed brain surface. The presurgical step is intended to minimize surgical trauma on the day of experimentation.
Chronic preparations in uninfected mice exhibited remarkably improved stability over acute ones by significantly reducing periarteriolar tissue damage and enhancing cerebroarteriolar dilator responses. The chronic scheme was successfully implemented in ECM mice which unveiled novel preliminary insights on impaired cerebroarteriolar reactivity and eNOS dysfunction.
The chronic scheme presents an innovative approach for advancing our mechanistic understanding on cerebrovascular dysfunction in ECM.
cerebroarteriolar responses; cranial window superfusion; endothelial nitric oxide synthase; experimental cerebral malaria; Plasmodium berghei ANKA
We examined insulin's uptake and transendothelial transport (TET) by cultured bovine aortic endothelial cells (BAECs) in order to: a) ascertain whether insulin accumulates within the cells to concentrations greater than in the media; b) compare the TET of insulin to that of inulin (using the latter as a tracer for passive transport or leak); and c) determine whether insulin's TET depended on insulin action. Using 125I-insulin at physiologic concentrations, we found that BAECs accumulate insulin >5-fold above media concentrations and that the TET of insulin, but not inulin, is saturable and requires intact PI-3-kinase and MEK-kinase signaling. We conclude that the insulin receptor and downstream signaling from the receptor regulate endothelial insulin transport. Based on comparison of the kinetics of BAEC insulin uptake with insulin TET, we suggest that insulin uptake is rate limiting for insulin TET.
A clinical measure of endothelial glycocalyx structure would have great potential importance because lesions of the glycocalyx may be the first changes to occur in diabetes and in a wide range of vascular diseases. A method recently described by Nieuwdorp et al [15, 16] for estimating the volume of the luminal glycocalyx of the entire human vascular system would seem to be the first attempt to develop a measure of this kind. It is based on the tracer dilution principle and this review considers the principles and conditions that underlie this method and the extent to which the conditions appear to have been fulfilled in this case. Our analysis raises two questions about (a) the estimation of the concentration of the tracer (dextran 40) at zero time and (b) the estimation of plasma volume, both of which can be answered by changes in experimental protocol. A third question, concerning the partition coefficient of the tracer between plasma and the fluid within the glycocalyx, cannot be answered at the present time and until it has been resolved, glycocalyx volume cannot be estimated from the dilution of a macromolecular tracer.
We sought to determine some of the molecular requirements for basal state “tone” of skeletal muscle arterioles in vivo, and whether asynchronous Ca2+ waves are involved or not.
Cremaster muscles of anesthetized exMLCK and smGCaMP2 biosensor mice were exteriorized, and the fluorescent arterioles were visualized with wide-field, confocal or multiphoton microscopy to observe Ca2+ signaling and arteriolar diameter.
Basal state tone of the arterioles was~50%. Local block of Ang-II receptors (AT1) or α1-adrenoceptors (α1-AR) had no effect on diameter, nor did complete block of sympathetic nerve activity (SNA). Inhibition of phospholipase C caused dilation nearly to the Ca2+-free (passive) diameter, as did exposure to nifedipine or 2-APB. Arterioles were also dilated when treated with SKF96365. High-resolution imaging of exMLCK fluorescence (ratio) or GCaMP2 fluorescence in smooth muscle cells failed to reveal Ca2+ waves (although Ca2+ waves/transients were readily detected by both biosensors in small arteries, ex vivo).
Arterioles of cremaster muscle have vascular tone of ~50%, which is not due to α1-AR, AT1R, or SNA. PLC activity, L-type Ca2+ channels, 2-APB- and SKF96365-sensitive channels are required. Propagating Ca2+ waves are not present. A key role for PLC and InsP3R in vascular tone in vivo, other than producing Ca2+ waves, is suggested.
calcium; vascular tone; smooth muscle; arterioles; sympathetic nerve activity
Arterial tone is dependent on the depolarizing and hyperpolarizing currents regulating membrane potential and governing the influx of Ca2+ needed for smooth muscle contraction. Several ion channels have been proposed to contribute to membrane depolarization, but the underlying molecular mechanisms are not fully understood. In this review, we will discuss the historical and physiological significance of the Ca2+-activated cation channel, TRPM4, in regulating membrane potential of cerebral artery smooth muscle cells. As a member of the recently described transient receptor potential super family of ion channels, TRPM4 possesses the biophysical properties and upstream cellular signaling and regulatory pathways that establish it as a major physiological player in smooth muscle membrane depolarization.
Cerebral Artery Smooth Muscle Cells; TRPM4; Membrane Potential
The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of cerebral blood flow, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of parenchymal arterioles. There are, moreover, notable functional differences between pial arteries and parenchymal arterioles. For example, in pial vascular smooth muscle cells (VSMCs), local calcium release events (“calcium sparks”) through ryanodine receptor (RyR) channels in sarcoplasmic reticulum membrane activate large conductance, calcium-sensitive potassium (BK) channels to modulate vascular diameter. In contrast, VSMCs in parenchymal arterioles express functional RyR and BK channels, but under physiological conditions these channels do not oppose pressure-induced vasoconstriction. Here we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of cerebral blood flow.
brain parenchymal arteriole; cerebral blood flow; acidosis; ryanodine receptor; potassium channel
Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells. These signals vary with respect to their mechanisms of generation, temporal properties, and spatial distributions. The calcium signals discussed include calcium waves, junctional calcium transients, calcium sparks, calcium puffs, and L-type calcium channel sparklets. For each calcium signal we address underlying mechanisms, general properties, physiological importance, and regulation.
This review addresses the latest advances in our understanding of the regulation of a novel Ca2+ signal called L-type Ca2+ channel sparklets in arterial smooth muscle. L-type Ca2+ channel sparklets are elementary Ca2+ influx events produced by the opening of a single or a small cluster of L-type Ca2+ channels. These Ca2+ signals were first visualized in the vasculature in arterial smooth muscle cells. In these cells, L-type Ca2+ channel sparklets display two functionally distinct gating modalities that regulate local and global intracellular Ca2+ concentration ([Ca2+]i). The activity of L-type Ca2+ channel sparklets varies regionally within a cell depending on the dynamic activity of a cohort of protein kinases and phosphatases recruited to L-type Ca2+ channels in the arterial smooth muscle sarcolemma in a complex coordinated by the scaffolding molecule A kinase anchoring protein 150 (AKAP150). We also described a mechanism whereby clusters of L-type Ca2+ channels gate cooperatively to amplify intracellular Ca2+ signals with likely pathological consequences.
Store-operated Ca2+ entry (SOCE) is a receptor-regulated Ca2+ entry pathway that is both ubiquitous and evolutionarily conserved. SOCE is activated by depletion of intracellular Ca2+ stores through receptor-mediated production of inositol 1,4,5-trisphosphate (IP3). The depletion of endoplasmic reticulum (ER) Ca2+ is sensed by stromal interaction molecule 1 (STIM1). On store depletion, STIM1 aggregates and moves to areas where the ER comes close to the plasma membrane (PM; within 25nm) to interact with Orai1 channels and activate Ca2+ entry. Ca2+ entry through store-operated Ca2+ (SOC) channels, originally thought to mediate the replenishment of Ca2+ stores, participate in active downstream signaling by coupling to the activation of enzymes and transcription factors that control a wide variety of long-term cell functions such as proliferation, growth and migration. SOCE has also been proposed to contribute to short-term cellular responses such as muscle contractility. While there are significant STIM1/Orai1 protein levels and SOCE activity in adult skeletal muscle, the precise role of SOCE in skeletal muscle contractility is not clear. The dependence on SOCE during cardiac and smooth muscle contractility is even less certain. Here, we will hypothesize on the contribution of SOCE in muscle and its potential role in contractility and signaling.