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Neurofibromatosis 1 (NF1) is an autosomal dominant disorder with complete penetrance and an estimated prevalence of 1 in 3000. Clinical manifestations are extremely variable. The disease is characterized by multiple café-au-lait spots (skin pigmentation), iris harmartomas, and multiple benign and malignant nerve sheath tumors (neurofibromas) in the central and peripheral nervous systems (Jett and Friedman 2010). NF1 vasculopathy is a serious and well documented but poorly understood feature of NF1. NF1 vasculopathy may result in hypertension, cerebrovascular disease, ischemia, aneurysm, hemorrhage or death in young adults (Rasmussen et al 2001). The response to standard therapeutic interventions is often disappointing (Friedman et al 2002), so further understanding of the underlying mechanisms for vascular complications in NF1 is essential. Endothelial and vascular smooth muscle functions are altered in Nf1+/- mice in vitro (Li et al 2001, Li et al 2006, Munchhof et al 2006), however, it is unknown how these alterations affect function of the vessel. We have compared vascular function of the thoracic aorta in Nf1+/- mice with age-matched control littermates (n=16) using a small vessel myograph (A/S Danish Myotechnology, Aarthus N, Denmark). Isometric force measurements revealed increased contraction in response to depolarization (maximal response of 3.3 ± 0.32 in Nf1+/- aorta versus 2.0 ± 0.22 in controls; p= 0.005), but diminished contraction in response to phenylephrine in Nf1+/- thoracic aorta at 6 months of age (pEC50 = 5.36 ± 0.18 in Nf1+/- aorta versus 7.02 ± 0.09 in controls; maximal response, Emax = 67.28 ± 3.4 in Nf1+/- aorta versus 86.34 ± 5.7; p= 0.008). Stress-strain curves indicated that arterial stiffness was increased at 6 months of age in Nf1+/-vessels. Elastin staining revealed disorganization of elastic fibers in Nf1+/- vessels. In conclusion, the pathogenesis of NF1 vasculopathy in the thoracic aorta increases stiffness and impairs vasomotor function. [ABSTRACT FROM AUTHOR]

We investigate a hypothesis for the generation of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ waves in the vascular smooth muscle of the pulmonary artery. Agonist-stimulated waves of elevated cytoplasmic calcium concentration regulate blood vessel tone and vasomotion in vascular smooth muscle. In rat pulmonary artery smooth muscle cells, the calcium mobilizing messenger NAADP appears to trigger bursts of Ca2+ release from lysosomal Ca2+ stores by activating the Two Pore Segment Channel subtype 2 (TPC2). It is suggested that these Ca2+ transients initiate a propagating wave by Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Deconvolution and confocal microscopy observations, including immunofluorescence, indicate that lysosome clusters may selectively couple to RyR subtype 3 (RyR3) in regions where lysosomes and proximal SR are separated by a narrow space possibly < 100 nm and beyond the resolution of light microscopy. These results naturally lead to the hypothesis that lysosome-SR (L-SR) junctions may form a cytoplasmic trigger zone for the observed Ca2+ bursts and subsequent cell-wide Ca2+ waves. The present study combines prior optical microscopy observations with a thorough ultrastructural characterization of the L-SR junctions in rat pulmonary artery smooth muscle as input data for a quantitative model of the L-SR junction to test the above hypothesis. With this model, we simulate the Ca2+ bursts that may be generated in the LSR junctions to determine whether or not these bursts give rise to a sufficient increase in junctional [Ca2+] to breach the threshold for RyR3 activation by CICR and thus initiation of a propagating Ca2+ wave. We are grateful to Garnet Martens and the University of British Columbia Bioimaging Facility for their guidance in electron microscopy imaging, to David Walker for his insightful help with ultrastructure identification, to Tom Bartol for his continual assistance with MCell, and to Arash Tehrani for helping with image analysis. This work was supported by Grant No. CIHR MOP-84309 from the Canadian Institute of Health Research and by the British Heart Foundation Project Grants PG/03/065 and PG/10/95/28657. [ABSTRACT FROM AUTHOR]

Physiological selection between different modes of cell function is based on the segregation of Ca2+ transients in different locations of the cytoplasm. Evidence in the literature suggests that in smooth muscle such segregation is effected by a large variety of specialized SR-organelle nano-junctions, each controlling the [Ca2+] in a nano-space near Ca2+ sensitive enzymes, channels, pumps and exchangers (Pan-Junctional SR). If different parts of the Pan-Junctional SR generate different localized [Ca2+], then it is likely that the SR itself is compartmentalized. However, it has been shown that the SR luminal Ca2+ concentration can be independently regulated via PM-SR junctions and that there is structural continuity throughout the SR and nuclear envelope. The answer to this paradox could be resolved by assuming that the SR lumen is irregular, has spatially separated clusters of sarco/endoplasmic reticulum Ca2+ ATPases (SERCA), inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR) and exhibits areas of restricted diffusion. Therefore, the nanospaces bordering both surfaces of the SR are critical in defining the specificity of the Ca2+ signals. In this presentation, we propose that the typical make-up of the Pan-junctional SR determines the smooth muscle type and pose the question of how to formulate a quantitatively testable hypothesis describing Ca2+ transport mechanism in junctions between the membranes of the SR and other organelles, including the plasma-membrane (PM). All vascular smooth muscles respond to physiological stimulation with Ca2+ oscillations, but three different types of Ca2+ oscillations have been reported for various blood vessels: 1. Waves of Ca2+ induced Ca2+ release (CICR) involving IP3R initiated by elevation of IP3. 2. Waves of CICR involving RyR, initiated by nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated lysosomal Ca2+ release. 3. Non wave-like Ca2+ oscillations paced by periodic SR Ca2+ release. Since Ca2+ release channels exhibit Ca2+ sensitivity on both their cytoplasmic and luminal terminals, fluctuations in regional luminal Ca2+ can also function as pacemakers to determine the frequency of SR Ca2+ release waves and that of nonwave- like Ca2+ oscillations. How do we test these provocative hypotheses? At present, specific hypotheses borne out of experimental observations often yield only cartoon models and can hardly be verified by conventional experimental methods. We argue that realistic quantitative modeling of the stochastic processes characterizing ion transport in the junctions is a necessary tool on the one hand for testing of such hypothesis and on the other to generate further testable predictions on SR junction mechanisms. We briefly overview a quantitative modeling approach that integrates the available experimental evidence into a realistic reproduction of two specific vascular smooth muscle SR junctions: (1) the PM-SR junction, which appears to be at the base of CICR wave phenomena originating from IP3R Ca2+ release from the SR upon adrenergic stimulation; (2) the lysosome-SR junction, in which NAADP-mediated Ca2+ release from two pore segment channels type 2 (TPC2) seems to originate Ca2+ bursts, which cause cell-wide CICR at RyR. Finally, we address the general question of how this approach could further our understanding of the coordinated regulation of such diverse processes as vaso-motor tone, metabolism and nuclear transcription. [ABSTRACT FROM AUTHOR]

Stromal interaction molecule (STIM1) and its isoform (STIM2) are single span sarco/endoplasmic (SR) transmembrane proteins that function as powerful SR Ca2+ sensors. When the SR Ca2+ content decreases STIM proteins migrate in proximity of the plasma membrane to tether and activate the Orai channels initiating the so called store operated Ca2+ entry (SOCE). In non-excitable cells STIM mediates Ca2+ entry that is required for regulating cell proliferation and migration. Smooth muscle cells (SMC) can exist as non-excitable cells, known also as the "proliferative" phenotype, or as excitable cells, known as the "contractile" phenotype. Furthermore, SMC can interchange their phenotype in response to environmental stimuli; Ca2+ signaling plays a crucial role in regulating this transition. However, very little is known about the role of STIM in SMC. Because isolated primary SMC quickly lose their contractile phenotype when placed in culture, the role of STIM proteins in SMC has been eluded. To overcome this limitation we used the Cre-lox technology approach to generate SM-specific STIM1-, STIM2-, and STIM1/STIM2- knockout (KO) mice, this model allowed us to systematically analyze the physiological role of STIM in SMC. SM-STIM1-KO mice survival rate was only about 50% within the first 30 days after birth. In addition, SM-STIM1-KO mice showed a consistent reduced body weight when compared to control mice. While the SM-STIM1/STIM2 double-KO phenotype was perinatally lethal, the SM-STIM2-KO was without a detectable phenotype. However, in the SM-STIM1 KO mice the STIM2 expression is enough to rescue the otherwise lethal phenotype, revealing that also STIM2 plays an important role in the SMC. Smooth muscle containing organs, such as intestine and aorta harvested from SM-STIM1-KO mice revealed morphological abnormalities when compared with organs harvested from control mice. Vascular reactivity analyzed using wire myography revealed that while depolarization-induced aortic contraction was unchanged, phenylephrine-mediated contraction was reduced by 26%, and store-dependent contraction was almost eliminated in aortas isolated from SM-STIM1-KO mice. Neointima formation induced by partial carotid artery ligation was suppressed by 54%. Consistently, in vitro PDGF-induced SMC proliferation was also reduced by 79% in STIM1-KO SMC. Notably, the Ca2+ store-refilling rate in STIM1-KO SMCs was substantially reduced, and sustained PDGF-induced Ca2+ entry was abolished. This defective Ca2+ homeostasis prevents PDGF-induced NFAT activation in both contractile and proliferating SMCs. In conclusion, our data show that STIM1-regulated Ca2+ homeostasis is crucial for NFAT-mediated transcriptional control required for induction of SMC proliferation, development, and growth during physiological as well as pathophysiological conditions. [ABSTRACT FROM AUTHOR]

Acidosis is a powerful vasodilator signal in the brain circulation. However, the mechanisms by which this response occurs are not well understood, particularly in the cerebral microcirculation. Ryanodine receptors (RyRs) are Ca2+ permeable channels in the sarcoplasmic reticulum. They dilate cerebral (pial) arteries by activation of large-conductance, calcium-sensitive potassium (BKCa) channels by local Ca2+ signals (Ca2+ sparks). This mechanism is clearly demonstrated by the non-additive constrictions induced by RyR or BKCa channel blockers in pressurized pial arteries. Nevertheless, these blockers have little effect on the diameter of pressurized parenchymal arterioles (PAs) from the brain, even though functional BKCa channels and RyRs are present. To determine the mechanism by which acidosis dilates brain PAs and to elucidate the roles of RyRs and BKCa channels in this response, internal diameter and vascular smooth muscle cell Ca2+ signals were measured in isolated pressurized murine PAs, using imaging techniques. At physiological pH (7.4) vascular smooth muscle cells exhibited largely Ca2+ waves but not Ca2+ sparks. Reducing external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca2+wave activity, and dramatically increased Ca2+ spark activity. Acidic pH caused a dilation of PAs which was inhibited by about 60% in a non-additive manner by BKCa channel blocker (1 µM paxilline) or RyR blocker (ryanodine 10 µM). Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BKCa channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of KATP channels (1 µM glibenclamide) or nitric oxide synthase (100 µM LNAME). These results support the novel concept that acidification, by converting Ca2+ waves to sparks, leads to the activation of BKCa channels to induce dilation of cerebral PAs. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension is driven by smooth muscle contraction and by the switch from a contractile to a migratory-proliferative smooth muscle phenotype(s), which requires changes in gene expression. These processes are selected, in part, by calcium signals, but how different calcium signals are generated to select each function is enigmatic. We have previously shown that SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM) in pulmonary arterial smooth muscle cells (PASMCs; Clark et al., 2010); i.e. to the superficial buffer barrier formed by PM-SR nanojunctions (van Breemen et al., 2013). Here they may support vasodilation in response to β-adrenoceptor activation. Induced increases in SERCA2b activity may thus remove calcium from the deeper cytoplasm to the superficial SR, from which calcium sparks may be released into the PM-SR junction via RyR1 to activate PM resident BKCa channels, driving hyperpolarisation (Boittin et al., 2003), removal of calcium from the cell by forward mode NCX activity and ultimately vasodilation (Boittin et al., 2003; Clark et al., 2010). In marked contrast, SERCA2a is entirely restricted to the deep, perinuclear SR (Clark et al., 2010) and may function to recycle calcium into this sub-compartment in support of vasoconstriction (Dipp & Evans, 2001; Dipp et al., 2001; Kinnear et al., 2004; Clark et al., 2010). Importantly, different subtypes of RyR are also strategically positioned here. RyR3 is preferentially targeted to the perinuclear SR and associated lysosome- SR nanojunctions (Kinnear et al., 2004, Kinnear et al., 2008), while the distribution of RyR2 is more widespread and extends from this region to the wider cell (Kinnear et al., 2008; Clark et al., 2010). We therefore proposed that perinuclear clusters of RyR3 may act as an initiation site for propagating calcium waves and contraction (Dipp & Evans, 2001; Kinnear et al., 2008). Thereafter, by calcium-induced calcium release, RyR2 may carry propagating calcium waves away from perinuclear clusters of RyR3 and across the myofilaments to enhance myocyte contraction (Kinnear et al., 2008; Clark et al., 2010). We have now identified a third subtype of SERCA pump in PASMCs, namely SERCA1, which together with RyR1 preferentially lines the nucleoplasmic reticulum (nSR). By contrast, we find no evidence of similar nSR labeling for other SERCA or RyR. It seems likely, therefore, that a variety of calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within and serve to demarcate different nanojunctions of the SR and their respective cytoplasmic nanodomains. Function-specific calcium signals may thus arise to provide for selective modulation of smooth muscle contraction and gene expression. [ABSTRACT FROM AUTHOR]